Biology subjects

Yildiz, A.

Publications and source records attributed to Yildiz, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Microtubule Lattice Spacing Governs MAP-Motor Regulation

Microtubules (MTs) serve as intracellular tracks that enable molecular motors to transport cargos to specific cellular destinations. It has been proposed that the signals directing motor-driven transport are encoded on MTs through different isotypes, lattice conformations, and post- translational modifications (PTMs) of tubulin, or MT-associated proteins (MAPs) that decorate the MT surface. However, molecular predictions of these models have not been rigorously tested in vitro. Using isotypically pure recombinant tubulin and biochemical reconstitution, we examined how tubulin PTMs and MT lattice spacing influence MAP binding and kinesin-1 motility. We found that kinesin-1 is largely insensitive to tubulin PTMs but is strongly regulated by MT lattice spacing. Likewise, the MAPs tau, MAP7, MAP4, DCX, and MAP9 exhibit little sensitivity to tubulin PTMs, whereas the MT-binding affinities of tau, DCX, and MAP7 depend on lattice spacing. In the presence of activating (MAP7) and inhibitory (tau) MAPs, lattice spacing determines MAP occupancy and thereby controls kinesin-1 motility. These findings support a two-layer transport code in which MT lattice spacing directs MAP binding, and MAPs determine which motors can move along individual MT tracks.

biochemistry↗

bioRxiv2026

A mechanism-aware stoichiometry platform resolves functional viral thresholds and induces antiviral hypersensitivity

Antiviral discovery is frequently hindered by a stoichiometric blind spot--a lack of resolution regarding the functional density of viral enzymes required for replication. Traditional screening pipelines rely on target-based or phenotypic assays that cannot distinguish between simple molecular binding and the crossing of a functional stoichiometric cliff. Here, we present a mechanism-aware platform that resolves these enzymatic requirements with single-virion precision. By integrating quantitative cryo-electron microscopy and genomic validation with Monte Carlo modeling, we map the stoichiometric landscapes of HIV-1 Protease (PR) and Reverse Transcriptase (RT). We uncover a striking disparity in enzymatic demand: a high-redundancy buffering capacity for PR ([~]40 monomers) contrasted with a high-threshold requirement for RT ([~]95 subunits). We demonstrate that by systematically de-buffering the virion, our platform induces a state of antiviral hypersensitivity, enabling the detection of therapeutic activity in novel and clinical inhibitors that remain invisible to traditional workflows. Furthermore, this multiplexed profiling enables de novo target identification, as inhibitors trigger failure exclusively on their respective stoichiometric arms. This platform provides a deterministic roadmap for de-risking drug discovery and identifying viral sub-stoichiometric vulnerabilities.

biophysics↗

bioRxiv2025

Structural Basis of Human Kinetochore-Microtubule Coupling by the Ndc80 and Ska Complexes

Faithful chromosome segregation depends on kinetochore-microtubule attachments that must be load-bearing, dynamic, and tightly regulated. In human cells, the Ska and Ndc80 complexes (SkaC and Ndc80C) cooperate to sustain these attachments, but how they are organized on microtubules and coupled to end dynamics remains unclear. Here, our cryo-EM structure shows that Ndc80C and SkaC form a coupled assembly across neighboring protofilaments. A phosphorylation-sensitive SKA3 tether binds a defined kink in the Ndc80 coiled-coil, positioning SKA1 beside Ndc80C. These two complexes jointly sandwich the -tubulin tail and sense its polyglutamylation state. SkaC recognizes the extended microtubule lattice and, through the SKA3 tether, confers this selectivity onto Ndc80C, enabling Ndc80C tip tracking while strengthening microtubule stabilization. Together, our findings define the structural basis of Ndc80C-SkaC cooperation and uncover a phosphorylation-regulated, polyglutamylation-sensitive, and lattice-state-dependent mechanism for robust and regulated KT-MT coupling during chromosome segregation in mitosis.

biophysics↗

bioRxiv2025

Structure and mechanism of microtubule stabilization and motor regulation by MAP9

Microtubule-associated proteins (MAPs) regulate the organization of microtubules and control intracellular transport, but their individual contributions to microtubule dynamics and motor regulation remain poorly understood. Here, we identify MAP9 as a critical factor that stabilizes microtubules and facilitates neuronal morphogenesis. MAP9 knockdown abolishes the outgrowth of neurites, a phenotype not observed through the loss of other neuronal MAPs. Cryo-electron microscopy revealed that, unlike other MAPs that bind along protofilaments, MAP9 binds around the microtubule as a long alpha helix using five consecutive repeats. This unique binding mode enables MAP9 to staple adjacent protofilaments, thereby preventing microtubule depolymerization. We also showed that MAP9 selectively permits kinesin-3 and dynein motility while hindering kinesin-1 through interactions with a divergent loop-8 of the kinesin motor domain. Our results establish MAP9 as a key MAP required for neuronal growth and uncover how it differentially regulates intracellular transport driven by kinesin motors.

biophysics↗

bioRxiv2025

Enhanced Processivity and Collective Force Production of Kinesins at Low Radial Forces

Kinesin-1 is a robust motor that carries intracellular cargos towards the plus ends of microtubules. However, optical trapping studies reported that kinesin-1 is a slippery motor that quickly detaches from the microtubule, and multiple kinesins are incapable of teaming up to generate large collective forces. This may be due to the vertical (z) forces that the motor experiences in a single bead trapping assay, accelerating the detachment of the motor from a microtubule. Here, we substantially lowered the z-force by using a long DNA handle between the motor and the trapped bead and characterized the motility and force generation of single and multiple kinesin-1s. Contrary to previous views, we show that kinesin-1 is a robust motor that resists microtubule detachment before it reaches high hindering forces, but it quickly detaches under assisting forces even at low z-forces. We also demonstrate highly efficient collective force generation by multiple kinesin-1 motors. These results provide an explanation for how multiple kinesins team up to perform cellular functions that require higher forces than a single motor can bear.

biophysics↗

bioRxiv2025

Covariance-Based MD Simulation Analysis Pinpoints Nanobody Attraction and Repulsion Sites on SARS-CoV-2 Omicron Spike Protein

Mutations in the SARS-CoV-2 spike receptor-binding domain (RBD) of the Omicron variant enable broad escape from neutralizing antibodies (Abs) and nanobodies (Nbs), yet the atomistic basis of epitope-dependent loss of Nb binding remains unclear. We performed all-atom molecular dynamics (MD) simulations of 13 Nbs bound to the Omicron RBD to characterize their binding modes, binding-pose stability, and interfacial dynamics. Analysis of the trajectories and corresponding free-energy landscapes revealed that most Nbs occupied a single dominant basin but showed varying degrees of within-basin heterogeneity and often exhibited orientation shifts, whereas two Nbs (Nb21 and Sb14) showed pronounced pose plasticity and dissociated from the RBD. For the remaining 11 Nbs, covariance-matrix analysis mapped stabilizing and destabilizing residue couplings, revealing recurrent hydrophobic anchor patches within the receptor-binding motif and Nb-specific interaction patterns reflecting differences in the sequences and chemistries of their complementarity-determining regions. Omicron substitutions rewired interaction networks, shifted Nb binding orientations relative to the corresponding experimentally determined reference structures (obtained with the ancestral WT RBD for all Nbs except H3), and introduced unfavorable repulsion, either directly at mutation sites or indirectly via mutation-driven reorientation, thereby reducing interfacial stability. Low-speed steered MD unbinding simulations of the 11 Nb-RBD complexes and the ACE2-RBD complex, performed at a pulling velocity within the range used in high-speed AFM-based force spectroscopy, yielded lower mean unbinding work for all 11 Nbs than for ACE2. These simulations also revealed heterogeneous, stepwise Nb unbinding pathways in which receptor-binding ridge (RBR)-mediated anchoring maintained partial Nb attachment through newly formed interactions and stabilized transient intermediate binding poses. Together, these Nb-RBD interaction fingerprints and pathway-resolved unbinding simulations pinpoint epitope-specific determinants and mutation-induced clash sites, providing a mechanistic basis for diminished Nb binding.

biophysics↗

bioRxiv2024

Nde1 Promotes Lis1 Binding to Full-Length Autoinhibited Human Dynein-1

Cytoplasmic dynein-1 (dynein) is the primary motor for the retrograde transport of intracellular cargoes along microtubules. The activation of the dynein transport machinery requires the opening of its autoinhibited Phi conformation by Lis1 and Nde1/Ndel1, but the underlying mechanism remains unclear. Using biochemical reconstitution and cryo-electron microscopy, we show that Nde1 significantly enhances Lis1 binding to autoinhibited dynein and facilitates the opening of Phi. We discover a key intermediate step in the dynein activation pathway where a single Lis1 dimer binds between the Phi-like (PhiL) motor rings of dynein. In this "PhiL-Lis1", Lis1 interacts with one of the motor domains through its canonical interaction sites at the AAA+ ring and stalk and binds to the newly identified AAA5, AAA6, and linker regions of the other motor domain. Mutagenesis and motility assays confirm the critical role of the PhiL-Lis1 interface. This intermediate state is instantly and efficiently formed in the presence of Nde1, but Nde1 is not part of the PhiL-Lis1. These findings provide key insights into the mechanism of how Nde1 promotes the Lis1-mediated opening of Phi dynein.

biophysics↗

bioRxiv2024

NuMA mechanically reinforces the spindle independently of its partner dynein

Both motor and non-motor proteins organize microtubules to build the spindle and maintain it against opposing forces. NuMA, a long microtubule binding protein, is essential to spindle structure and function. NuMA recruits the motor dynein to spindle microtubule minus-ends to actively cluster them, but whether NuMA performs other spindle roles remains unknown. Here, we show that NuMA acts independently of dynein to passively reinforce the mammalian spindle. NuMA that cannot bind dynein is sufficient to protect spindle poles against fracture under external force. In contrast, NuMA with a shorter coiled-coil or disrupted self-interactions cannot protect spindle poles, and NuMA turnover differences cannot explain mechanical differences. In vitro, NuMAs C-terminus self-interacts and bundles microtubules without dynein, dependent on residues essential to pole protection in vivo. Together, this suggests that NuMA reinforces spindle poles by crosslinking microtubules, using its long coiled-coiled and self-interactions to reach multiple, far-reaching pole microtubules. We propose that NuMA acts as a mechanical "multitasker" targeting contractile motor activity and separately crosslinking microtubules, both functions synergizing to drive spindle mechanical robustness.

cell biology↗

bioRxiv2024

Structural and functional insights into activation and regulation of the dynein-dynactin-NuMA complex

During cell division, NuMA orchestrates the focusing of microtubule minus-ends in spindle poles and cortical force generation on astral microtubules by interacting with dynein motors, microtubules, and other cellular factors. Here, we used in vitro reconstitution, cryo-electron microscopy, and live cell imaging to understand the mechanism and regulation of NuMA. We determined the structure of the processive dynein/dynactin/NuMA complex (DDN) and showed that the NuMA N-terminus drives dynein motility in vitro and facilitates dynein-mediated transport in live cells. The C-terminus of NuMA directly binds to and suppresses the dynamics of the microtubule minus-end. Full-length NuMA is autoinhibited for its interactions with dynein and microtubules, but mitotically phosphorylated NuMA activates dynein in vitro and interphase cells. Together with dynein, activated full-length NuMA focuses microtubule minus-ends into aster-like structures. These results provide critical insights into the activation of NuMA and dynein for their mitotic functions.

biophysics↗

bioRxiv2024

Protection of the Telomeric Junction by the Shelterin Complex

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging between the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest to the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared to POT1 alone by bridging single- and double-stranded telomeric tracts. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/608453v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@a58e79org.highwire.dtl.DTLVardef@12cb172org.highwire.dtl.DTLVardef@135bf40org.highwire.dtl.DTLVardef@19f3fe4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Stepping dynamics of dynein characterized by MINFLUX

Cytoplasmic dynein is the principal motor for minus-end-directed motility and force generation functions along microtubules (MTs)1. Dynein converts the chemical energy of ATP hydrolysis into coordinated structural changes to step processively along MTs, but how dynein couples ATP hydrolysis to a minus-end-directed step remains controversial2, 3. The dynamics of dynein stepping have previously been characterized by tracking flexible regions of the motor with limited resolution4-6. Here, we site-specifically labeled yeast dynein at its MT-binding domain by developing a cysteine-light mutant and tracked its stepping at sub-millisecond and nanometer resolution at physiological ATP concentrations using MINFLUX7. We show that dynein hydrolyzes one ATP per step and takes multiples of 8 nm steps. Steps are preceded by a transient movement towards the plus end. These backward "dips" correspond to MT release upon ATP binding and subsequent diffusion of the stepping monomer around its MT-bound partner. Functional assays showed that dips terminate with a minus-end-directed movement upon ATP hydrolysis. These results provide critical insights into the order of mechanochemical events that result in a productive step of dynein.

biophysics↗

bioRxiv2024

Molecular interplay between HURP and Kif18A in mitotic spindle regulation

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determined the binding mode of HURP to microtubules using Cryo-EM. The structure reveals that one HURP motif spans laterally across {beta}-tubulin, while a second motif binds between adjacent protofilaments. HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observed that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in spindle length control.

biophysics↗

bioRxiv2024

A unique and biocompatible approach for corneal collagen crosslinking in vivo

Corneal crosslinking (CXL) is a widely applied technique to halt the progression of ectatic diseases by increasing the thickness and mechanical stiffness of the cornea. This study investigated the biocompatibility and efficiency of a novel CXL procedure using ruthenium and blue light in rat corneas and evaluated factors important for clinical application. To perform the CXL procedure, the corneal epithelium of rats was removed under anesthesia, followed by the application of a solution containing ruthenium and sodium persulfate (SPS). The corneas were then exposed to blue light at 430 nm at 3 mW/cm2 for 5 minutes. Rat corneas were examined and evaluated for corneal opacity, corneal and limbal neovascularization, and corneal epithelial regeneration at days 0, 1, 3, 6, 8, and 14. On day 28, the corneas were isolated for subsequent tissue follow-up and analysis. CXL with ruthenium and blue light showed rapid epithelial healing, with 100% regeneration of the corneal epithelium and no corneal opacity by day 6. The ruthenium group also exhibited significantly reduced corneal (p<0.01) and limbal neovascularization (p<0.001). Histological analysis revealed no signs of cellular damage or apoptosis, which further confirms the biocompatibility and nontoxicity of our method. Confocal and scanning electron microscopy (SEM) images showed a greater density of collagen fibrils, indicating efficient crosslinking and enhanced structural integrity. This study confirmed the in vivo safety, biocompatibility, and functionality of ruthenium and blue light CXL. This method can prevent toxicity caused by UV-A light and can be a rapid alternative treatment to standard crosslinking procedures. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/585574v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@c05147org.highwire.dtl.DTLVardef@18244borg.highwire.dtl.DTLVardef@f6e30eorg.highwire.dtl.DTLVardef@b4ec3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Torques within and outside the human spindle balance twist at anaphase

At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, using lattice light sheet microscopy, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are redundantly required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.

cell biology↗

bioRxiv2023

A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression

The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dyneins diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dyneins well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

cell biology↗

bioRxiv2023

The Mechanism and Energetics of the Dynein Priming Stroke

Dyneins is an AAA+ motor responsible for motility and force generation towards the minus end of microtubules. Dynein motility is powered by nucleotide-dependent transitions of its linker domain, which transitions between straight (post-powerstroke) and bent (pre-powerstroke) conformations. To understand the dynamics and energetics of the linker, we per-formed all-atom molecular dynamics (MD) simulations of human dynein-2 primed for its power stroke. Simulations re-vealed that the linker can adopt either a bent conformation or a semi-bent conformation, separated by a 5.7 kT energy bar-rier. The linker cannot switch back to its straight conformation in the pre-powerstroke state due to a steric clash with the AAA+ ring. Simulations also showed that an isolated linker has a free energy minimum near the semi-bent conformation in the absence of the AAA+ ring, indicating that the linker stores mechanical energy as it bends and releases this energy during the powerstroke.

biophysics↗

bioRxiv2023

Nde1 is a Biphasic Regulator of the Dynein Activation Pathway

Cytoplasmic dynein is the primary motor that drives the motility and force generation functions towards the microtubule minus end. The activation of dynein motility requires its assembly with dynactin and a cargo adaptor. This process is facilitated by two dynein-associated factors, Lis1 and Nde1/Ndel1. Recent studies proposed that Lis1 rescues dynein from its autoinhibited conformation, but the physiological function of Nde1/Ndel1 remains elusive. Here, we investigated how human Nde1 and Lis1 regulate the assembly and subsequent motility of the mammalian dynein/dynactin complex using in vitro reconstitution and single molecule imaging. We found that Nde1 promotes the assembly of active dynein complexes in two distinct ways. Nde1 competes with the 2 subunit of platelet activator factor acetylhydrolase (PAF-AH) 1B, which recruits Lis1 as a noncatalytic subunit and prevents its binding to dynein. Second, Nde1 recruits Lis1 to autoinhibited dynein and promotes Lis1-mediated assembly of dynein-dynactin-adaptor complexes. However, excess Nde1 inhibits dynein, presumably by competing against dynactin to bind the dynein intermediate chain. The association of dynactin with dynein triggers Nde1 dissociation before the initiation of dynein motility. Our results provide a mechanistic explanation for how Nde1 and Lis1 synergistically activate the dynein transport machinery.

biophysics↗

bioRxiv2022

Lis1 binding regulates force-induced detachment of cytoplasmic dynein from microtubules

Cytoplasmic dynein-1 (dynein) is an AAA+ motor that transports intracellular cargos towards the microtubule minus end. Lissencephaly-1 (Lis1) binds to the AAA+ ring and stalk of dyneins motor domain and promotes the assembly of active dynein complexes. Recent studies showed that Lis1 slows motility when it remains bound to dynein, but the underlying mechanism remained unclear. Using single-molecule and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro. We showed that Lis1 does not slow dynein motility by serving as a roadblock or tethering dynein to microtubules. Lis1 binding also does not affect the forces that stall dynein movement, but it induces prolonged stalls and reduces the asymmetry in the force-induced detachment of dynein from microtubules. The mutagenesis of the Lis1 binding sites on dyneins stalk partially recovers this asymmetry but does not restore dynein velocity. These results suggest that Lis1s interaction with the AAA+ ring is sufficient to result in slower movement and that Lis1s interaction with dyneins stalk slows force-induced detachment of dynein from microtubules.

biophysics↗