Biology subjects

Diez, S.

Publications and source records attributed to Diez, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural mechanism of nuclear membrane sealing by LEM2-ESCRT-III

In open mitosis, re-establishing nucleocytoplasmic compartmentalization requires the LEM2-ESCRT machinery to coordinate spindle clearance with sealing of the remaining nuclear envelope pores. The structural basis of this topologically unique and fundamental membrane-remodeling process is poorly understood. Here, we combine biochemical reconstitution, cryo-electron tomography, subtomogram averaging and large-scale molecular dynamics simulations to define the structural mechanism of nuclear membrane sealing. We structurally resolve that LEM2s winged-helix domain (WH) co-polymerizes with the ESCRT-II/III protein CHMP7 to form a membrane-bound scaffold whose geometry is progressively remodeled by downstream ESCRT-III proteins as it transitions from the flat membrane surrounding the pore towards the negatively curved membrane neck. In parallel, LEM2 positions its intrinsically disordered low-complexity domain within the pore, where condensation around spindle microtubules mechanically couples the membrane-ESCRT-LEM2 scaffold to the spindle and narrows the remaining diffusion path, restoring compartmentalization before membrane closure is complete. Remarkably, the LEM2-WH domain alone forms tightly constricted membrane tubes, coating the negatively curved inner surface, revealing an intrinsic membrane-remodeling activity of the receptor itself. Together, our work establishes a structural framework for how receptor-ESCRT co-polymerization, low complexity domain-mediated sealing and receptor-driven membrane remodeling guide nuclear-envelope pores from spindle-containing openings to terminal constriction and fusion.

molecular biology↗

bioRxiv2026

A neuroimmune IL-13 axis is associated with human enteric nervous system development

The mechanisms governing maturation of the human enteric nervous system (ENS) during early postnatal life remain poorly defined. Here, we characterize the transcriptomic, cellular, and functional landscape of the neonatal human ileum and identify a neuro-immune axis associated with ENS expansion. Using human tissue transcriptomics, flow cytometry, iPSC-derived enteric neural lineages, and single-cell interactome analyses, we show that the neonatal ileum is enriched for pro-neurogenic transcriptional programs and harbors a greater abundance of enteric neurons and glia than the adult tissue. T cells emerge as a predominant source of interleukin-13 (IL-13) in the neonatal gut, and enteric neurons express its receptor IL13RA1, enabling direct immune-to-neuron signaling. Functional experiments demonstrate that IL-13 enhances expression of key enteric neuronal markers in a concentration-dependent manner. In parallel, single-cell analyses identify enteric neurons as a major predicted source of macrophage migration inhibitory factor (MIF), with signaling directed toward T and NK cell populations, suggesting that the ENS actively shapes the immune environment it depends upon. Together, these findings support a model in which bidirectional neuro-immune communication establishes a pro-neurogenic niche during a critical window of ENS development. This work positions the neonatal immune system as an active contributor to ENS maturation and offers a new perspective on how neuro-immune crosstalk shapes intestinal development in early life.

neuroscience↗

bioRxiv2026

Programmable microactuators phase-lock cilia to local oscillatory flow

Hydrodynamic synchronization of motile cilia is essential for biological functions such as fluid transport, locomotion, and developmental patterning. It comprises the generation and the response to local flows in complex geometries. Besides their central role in physiology, direct experimental tests of ciliary responses to local flows at cellular length and time scales have remained elusive, largely due to the absence of tools capable of applying controlled, and localized flow stimuli. Here, we introduce programmable, nanometer-thin Ti/Pt microactuators that generate well-defined hydrodynamic forcing at biologically relevant frequencies while operating at biocompatible sub-Volt voltages. This platform is pioneering a controlled local hydrodynamic stimulation of individual motile cilia. We quantify the flow fields and forces produced by single microactuators using particle image velocimetry. Applying local oscillatory flows close to motile cilia of the green alga Chlamydomonas reinhardtii, we probe their dynamic response by quantifying phase-locking between cilia and microactuators. This quantification is aided by combining machine-learning-based image segmentation, oscillator phase reconstruction, and circular statistics. During actuation, we observe signatures of phase-locking: those include a reversible modulation of the fluctuations in phase-difference between cilium and actuator and a systematic shift in ciliary beating frequency. Beyond providing a bio-compatible and precise platform for local hydrodynamic stimulation, our approach establishes an experimental framework for directly testing theories of hydrodynamic synchronization and load adaptation in systems of motile cilia.

biophysics↗

bioRxiv2026

Programmable DNA Origami Caps for Site-Selective Functionalization of Microtubule Tips and Lattice Defects

Microtubules are central components of cytoskeletal transport systems and have been widely repurposed as active elements in motor-driven nanodevices. However, site-specific functionalization of stabilized microtubules remains a fundamental challenge, as the tubulin lattice presents chemically indistinguishable binding sites along its length. Here we report a strategy for selective end-functionalization of stabilized microtubules using DNA origami nanostructures. By coupling DNA origami to Fab fragments targeting acetylated -tubulin Lys40 within the microtubule lumen, and exploiting steric exclusion of the origami from the lattice interior, binding is confined to accessible sites at microtubule ends and lattice defects. Using a six-helix bundle origami as a minimal construct, we demonstrate selective tip labelling of gliding microtubules without perturbing kinesin-driven motility. The same structures additionally mark lattice defects, enabling dynamic visualization of defect sites during transport. Furthermore, we show that tip-bound origami can hybridize with complementary DNA strands to capture cargo from surfaces in motion, establishing programmable, end-specific loading. This approach introduces a generalizable route to spatially controlled functionalization of cytoskeletal filaments, enabling new capabilities in molecular transport, nanoscale assembly, and the study of microtubule integrity and repair.

bioengineering↗

bioRxiv2025

Practically Error-Free Junctions Enable Solving Large Instances of Exact Cover Problems Using Network-Based Biocomputation

Network-based biocomputing (NBC) presents an energy-efficient, parallel computing approach for solving nondeterministic polynomial time (NP) complete problems by leveraging motor-driven cytoskeletal filaments that explore all possible solutions through nanofabricated networks in a massively parallel fashion. However, guiding errors at pass junctions, where filaments deviate from their intended path, currently limit the scalability of NBC systems. In this study, we addressed this critical challenge by fabricating sub-200 nm channel geometries using modified electron-beam-lithography and reactive-ion-etching protocols to physically constrain the trajectories of kinesin-driven microtubules and enhance path fidelity. Investigating junction designs with varying channel widths, we demonstrate that reducing channel width significantly lowers junction error rates. Practically error-free junction performance was achieved by scaling down the entire network geometry by a factor of two. These optimized junctions were incorporated into NBC networks that successfully solved 24- and 25-set instances of the Exact Cover problem, representing solution spaces of approximately 16 million and 33 million, respectively. This work establishes a new benchmark in NBC performance and represents a computational scale far beyond what has been achieved in prior demonstrations.

bioengineering↗

bioRxiv2025

MAP65-1/PRC1 reinforces microtubules through nucleation

Microtubules are dynamic cytoskeletal filaments that help shape cells and guide their response to external cues, including mechanical stress. How cells reorganize and reinforce microtubule arrays under stress remains poorly understood. Here, we show that MAP65-1/PRC1 promotes microtubule nucleation on pre-existing lattices in vitro, particularly on microtubules with lattice defects and damage sites associated with mechanical stress. MAP65-1 preferentially binds bent microtubules both in vitro and in cells, likely because bending induces lattice damage, enhancing nucleation on and bundling of these microtubules in vitro. This creates a potential feedback loop where mechanical stress promotes MAP65-1 binding, which in turn stabilizes microtubules by nucleation and bundling and reinforces the alignment of the array with mechanical stress. Thus, we reveal a previously unknown role for MAP65/PRC1 proteins in lattice-based nucleation and suggest a mechanism by which cells record and respond to mechanical stress through microtubule reorganization.

cell biology↗

bioRxiv2025

Kinesin-induced buckling reveals the limits of microtubule self-repair

Microtubules are stiff cytoskeletal polymers whose ability to rapidly switch between growth and disassembly relies on a metastable lattice. This metastability is also reflected in their sensitivity to environmental conditions and in intrinsic lattice dynamics, where spontaneous tubulin loss is balanced by tubulin incorporation from solution - a process that also enables microtubules to self-repair when damaged. Whether such intrinsic self-repair is sufficient to preserve microtubule integrity during dynamic molecular-motor induced buckling, which frequently occurs in cells, remains unclear. Here, we show that kinesin-driven microtubule buckling in vitro induces severe lattice damage, leading to extensive tubulin incorporation. In many cases, however, the damage exceeds the microtubules capacity for self-repair, resulting in breakage. In contrast, microtubules survive continuous buckling substantially longer in the presence of intracellular factors. Our results identify the limits of intrinsic microtubule self-repair and demonstrate that additional cellular mechanisms are essential to maintain microtubule integrity under sustained mechanical load.

biophysics↗

bioRxiv2025

Microtubule lattice defects facilitate spastin-mediated severing

The length regulation of microtubules and their organization into complex arrays inside cells occurs through the activity of polymerases, depolymerases as well as severing enzymes such as katanin and spastin. The latter hexamerize on the microtubule lattice, pull out single tubulin dimers in an ATP-dependent manner and eventually generate internal breaks in the microtubule. While both, katanin and spastin, were shown to be regulated by posttranslational tubulin modifications, only katanin was reported to have microtubule lattice-defect- or crossover-sensing activity. Here, we employ in vitro assays to investigate the impact of microtubule lattice defects on the severing characteristics of spastin. Toward this end, we prepared GMPCPP-stabilized microtubules with varying defect densities. Thereby, microtubule defects were introduced either through specific polymerization conditions or by end-to-end annealing of microtubules. We found that (i) the presence of defects accelerated the onset of the severing process and (ii) severing was twice as frequent in microtubule segments with defect sites as compared to random lattice segments. However, there was no evidence of preferential binding of spastin to defect sites. We therefore propose a severing mechanism in which defects do not actively promote microtubule severing but rather passively contribute to microtubule lattice instability, facilitating the process as fewer tubulin subunits are required to be removed for microtubule severing.

biophysics↗

bioRxiv2025

Altered lysosomal biology impairs motor neuron survival via TFEB dysregulation in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a devastating motor neuron disease, caused by recessive mutations or deletions of the SMN1 gene, representing the leading genetic cause of infant mortality. Available therapies, aimed at increasing SMN protein levels, can only partially halt motor neuron (MN) degeneration in a select number of patients, reinforcing the need for combinatorial treatments to improve clinical outcomes. We previously showed that mTORC1 overactivation and impaired autophagosome clearance in SMA MNs lead to the accumulation of protein aggregates, contributing to MN degeneration. However, the mechanistic link between SMN protein deficiency and autophagy-lysosomal dysfunction remained unknown. Here, using patient iPSC-derived MNs along with isogenic and healthy controls, we show that SMA MNs exhibit reduced lysosome numbers and impaired functionality. Furthermore, the master regulator of lysosomal biogenesis and autophagy, TFEB, is downregulated, and its nuclear translocation compromised upon SMN deficiency. We further propose the upregulation of the mTORC1 positive modulator TPT1 as contributor to TFEB dysregulation. Notably, TFEB overexpression ameliorates protein aggregate accumulation in SMA MNs and enhances MN survival both in vitro and in a zebrafish SMA model. Our findings identify lysosomal dysfunction as a key player in SMA pathology and highlight TFEB activation as a potential therapeutic strategy for SMA treatment. One Sentence SummaryTFEB activation restores lysosomal function and improves motor neuron survival in SMA, highlighting its potential as a therapeutic target.

neuroscience↗

bioRxiv2025

IQM-22110 as a selective KV4.3/KChIP3 modulator. Molecular determinants of the KChIP3 binding site

The goal of the present study was to discover novel KChIP ligands as research tools for modulating the KV4.3/KChIP channels. By employing a multidisciplinary approach, combining medicinal chemistry and electrophysiology studies, a novel KV4.3/KChIP modulator (IQM-22110) was successfully identified. IQM-22110 has emerged from the combination of our prior knowledge regarding the (phenylacetamido)benzoic acid moiety as an effective scaffold for KChIP3 ligands and a virtual screening of a focused chemical library. Guided by docking studies--which indicated that incorporating an additional aromatic ring could enhance binding affinity--IQM-22110 was selected for synthesis and identified as a potent KChIP3 ligand. Its electrophysiological effects on KV4.3/KChIP3 currents indicate that IQM-22110 binds to a high affinity site in KV4.3/KChIP3 channels that it is not present in KV4.3/KChIP2 or KV4.3. To the best of our knowledge, here we describe the first KChIP3 ligand that selectively modulates KV4.3/KChIP3 versus KV4.3/KChIP2 and KV4.3 alone channels. Given that KChIP2 is primarily expressed in heart, our findings might pave the way for the development of KV4.3/KChIP3 blockers with reduced cardiac side effects. Computational and site-directed mutagenesis studies allowed the identification of IQM-22110s binding site on KChIP3. Knowledge gained from our structural and functional studies with this novel KChIP3 ligand could establish the basis for drug discovery programs fostering treatments for diseases in which KV4.3/KChIPs channels are involved.

pharmacology and toxicology↗

bioRxiv2025

Dynamic switching of cell-substrate contact sites allows gliding diatoms to modulate the curvature of their paths

The directed motility of unicellular organisms is critical for their survival and ecological success, yet the mechanisms that enable rigid-walled cells to dynamically reorient and alter the shape of their trajectories remain poorly understood. Here, we investigate the gliding motility of Craspedostauros australis, a raphid pennate diatom that moves rapidly across submerged surfaces using an intracellular actomyosin motility complex and adhesive mucilage strand secretion. Using high-precision single-cell tracking, scanning electron microscopy (SEM), and interference reflection microscopy (IRM), we reveal how diatoms achieve diverse path curvatures by dynamically modulating the location of raphe-substrate contact and switching between one- and two-raphe contact gliding. Our results show that local curvature variations along the raphes dictate trajectory shapes, with one-raphe contact gliding producing highly curved paths, while two-raphe contact gliding results in paths of lower curvature. IRM imaging further confirms that transitions between these gliding modes underlie abrupt changes in path curvature and cell reorientation. This dynamic-raphe-switching mechanism is conserved across cell sizes and correctly predicts the increased path curvatures observed in smaller cells according to their more pronounced local raphe curvature. By quantitatively linking raphe geometry, cell-substrate attachment dynamics and motility patterns, our study provides insights into a novel motility mechanism that allows diatoms to adapt their movement to complex environments.

biophysics↗

bioRxiv2024

A floor-field model of tau-taxol competition explains tau envelope dynamics on taxol-stabilized microtubules

Tau is a microtubule-associated protein (MAP) that is highly expressed in neurons. Recent in-vitro studies have shown that tau molecules can segregate into cohesive envelopes on paclitaxel (taxol) stabilized microtubules. These envelopes act as selective barriers gating the motor transport and protecting microtubules against the action of severing enzymes. However, the mechanisms underlying the formation of these envelopes remain unclear. Recent in vitro reconstitution assays revealed that the formation of tau envelopes induces a compaction of the underlying microtubule lattice, whereas taxol binding induces lattice expansion, indicating competitive binding between tau and taxol on microtubules. In this study, we use physical modeling to investigate how this competition between tau and taxol regulates the formation of tau envelopes. Our model includes static and dynamic floor-fields, concepts adapted from pedestrian dynamics. By comparing our simulation results with experimental data, we demonstrate that tau-taxol competition on the microtubule lattice is sufficient to explain the features of the formation and disassembly of tau envelopes. Notably, tau-tau interactions are not necessary to account for the steadiness of these envelopes.

biophysics↗

bioRxiv2024

Tau accelerates tubulin exchange in the microtubule lattice

Microtubules are cytoskeletal filaments that exhibit dynamic tip instability and, as recent discoveries reveal, possess a dynamic lattice undergoing continuous tubulin loss and incorporation. In this study, we investigate the role of tau, a neuronal microtubule-associated protein (MAP) known for its stabilizing effects on microtubules, in modulating lattice dynamics. Using in vitro reconstitution, kinetic Monte Carlo modeling, and molecular dynamics simulations, we reveal that tau, despite lacking enzymatic activity, accelerates tubulin exchange within the lattice, particularly at topological defect sites. Tau appears to stabilize longitudinal tubulin-tubulin interactions while destabilizing lateral ones, thereby enhancing the mobility and repair of lattice defects. These results challenge the traditional view of tau as merely a stabilizer, uncovering its active role in dynamically modulating microtubule lattice structure.

biophysics↗

bioRxiv2024

Intellectual disability-causing mutations in KIF11 impair microtubule dynamics and dendritic arborization

Precise control of axonal and dendritic architecture is vital for proper brain function, with microtubule (MT) dynamics playing a central role in this process. Here, we uncover a previously unrecognized function of the molecular motor protein KIF11, which acts as a MT dynamics rheostat in hippocampal neurons to modulate dendritic branching. Known for its role in mitotic spindle bipolarity, KIF11 is also implicated in Microcephaly with or without chorioretinopathy, lymphedema, or intellectual disabilities (MCLID). However, the specific neuronal functions of KIF11 and the impact of its mutations in MCLID have remained largely unexplored. Our studies, using quantitative imaging of MT dynamics following KIF11 inhibition, indicate that KIF11 preferentially binds to parallel MTs in mature neurons. This binding is associated with a marked increase in minus-end-out MT dynamics in both axons and dendrites upon KIF11 loss of function, coupled with enhanced MT flux and extended growth in tertiary dendrites. These changes suggest a novel role for KIF11 in orchestrating dendritic branching. Moreover, introducing MCLID-associated KIF11 mutations, KIF11Y82F, and KIF11{Delta}Cterm, which cause minor microcephaly but severe intellectual disabilities, leads to significantly reduced MT dynamics and impaired dendritic arborization. In a microtubule sliding assay, KIF11Y82F significantly reduced KIF11 velocity while KIF11{Delta}Cterm increased it. Temporal inhibition of KIF11 using a photo-inhibitable KIF11, show increased MT dynamics and dendritic growth, while activation results in kinked and twisted branches. Together, these data reveal that KIF11 is MT dynamics rheostat and regulator of dendritic arborization in mature neurons and provide new insights into the molecular mechanisms driving MCLID.

neuroscience↗

bioRxiv2024

Tubulin tyrosination/detyrosination governs the spatial segregation of intraflagellar transport trains on axonemal microtubule doublets

Assembly and function of cilia rely on the continuous transport of ciliary components between the cell body and the ciliary tip. This is performed by specialized molecular machines, known as Intraflagellar Transport (IFT) trains. Anterograde IFT trains are powered by kinesin-2 motors and move along the B-tubules (enriched in detyrosinated tubulin) of the ciliary microtubule doublets. Conversely, retrograde IFT trains are moved by dynein-1b motors along the A-tubules (enriched in tyrosinated tubulin) back to the cell body. The segregation of oppositely directed trains on A-tubules or B-tubules is thought to prevent traffic jams in the cilium, but the mechanism by which opposite polarity trains are sorted onto either tubule, and whether tubulin tyrosination/detyrosination plays a role in that process, is unknown. Here, we show that CRISPR-mediated knock-out of VashL, the enzyme that detyrosinates microtubules, causes recurrent stoppages of IFT trains and reduces the rate of ciliary growth in Chlamydomonas reinhardtii. To test whether the observed stoppages, potentially caused by collisions between oppositely directed IFT trains, are ascribable to direct interactions between IFT trains and tubulin tyrosination/detyrosination, we developed methods to reconstitute the motility of native IFT trains from cilia on de-membranated axonemes and ex vivo on synthetically polymerized microtubules. We show that anterograde trains have higher affinity for detyrosinated microtubules (analogous to B-tubules), while retrograde trains for tyrosinated microtubules (analogous to A-tubules). We conclude that tubulin tyrosination/detyrosination is required for the spatial segregation of oppositely directed trains and for their smooth uninterrupted motion. Our results provide a model for how the tubulin code governs molecular transport in cilia.

cell biology↗

bioRxiv2024

Twist - torsion coupling in beating axonemes

Motile cilia and flagella are ubiquitous cell appendages whose regular bending waves pump fluids across tissue surfaces and enable single-cell navigation. Key to these functions are their non-planar waveforms with characteristic torsion. It is not known how torsion, a purely geometric property of the shape, is related to mechanical deformations of the axoneme, the conserved cytoskeletal core of cilia and flagella. Here, we assess torsion and twist in reactivated axonemes isolated from the green alga Chlamydomonas reinhardtii. Using defocused darkfield microscopy and beat-cycle averaging, we resolve the 3D shapes of the axonemal waveform with nanometer precision at millisecond timescales. Our measurements reveal regular hetero-chiral torsion waves propagating base to tip with a peak-to-peak amplitude of 22 {o}/{micro}m. To investigate if the observed torsion results from axonemal twist, we attach gold nanoparticles to axonemes to measure its cross-section rotation during beating. We find that locally, the axonemal cross-section co-rotates with the bending plane. This co-rotation presents the first experimental evidence for twist-torsion coupling and indicates that twist waves propagate along the axoneme during beating. Our work thus links shape to mechanical deformation of beating axonemes, informing models of motor regulation that shape the beat of motile cilia.

biophysics↗

bioRxiv2024

Gliding motility of the diatom Craspedostauros australis correlates with the intracellular movement of raphid-specific myosins

Raphid diatoms are one of the few eukaryotes capable of gliding motility, which is remarkably fast and allows for quasi-instantaneous directional reversals. Besides other mechanistic models, it has been suggested that an actomyosin system provides the force for diatom gliding. However, in vivo data on the dynamics of actin and myosin in diatoms are lacking. In this study we demonstrate that the raphe-associated actin bundles required for diatom movement do not exhibit a directional turnover of subunits and thus their dynamics do not contribute directly to force generation. By phylogenomic analysis we identified four raphid diatom-specific myosins in Craspedostauros australis (CaMyoA-D) and investigated their in vivo localization and dynamics through GFP-tagging. Only CaMyoB-D but not CaMyoA exhibited coordinated movement during gliding, consistent with a role in force generation. The characterization of raphid diatom-specific myosins lays the foundation for unraveling the molecular mechanisms that underlie the gliding motility of diatoms.

cell biology↗

bioRxiv2023

Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other

During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed rotation of anti-parallel microtubules around each other. The effective sidestepping probability of KIF11 increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbit without forward motion, with nearly full extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, enable flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.

biophysics↗