Biology subjects

Sept, D.

Publications and source records attributed to Sept, D..

12 recordsLinked to original sources

bioRxiv2026

Taxol exploits molecular switches within tubulin to stabilize microtubules.

Microtubules are dynamic filaments of tubulin heterodimers that comprise an essential part of the eukaryotic cytoskeleton1. The nucleotide state of tubulin controls microtubule dynamics: stable GTP-microtubules favor polymerization, whereas unstable GDP-microtubules drive depolymerization2. Anticancer compounds such as Taxol (paclitaxel) target microtubule dynamicity by preventing microtubule depolymerization3,4. Despite decades of work, the molecular basis of microtubule dynamics remains poorly defined. Using cryo-EM, we determined [~]2.2 [A] structures of human microtubules in GTP-like (GMPCPP) and GDP states. Comparison of these two states revealed switch-like structural changes as tubulins transition from the pre-hydrolysis (GMPCPP) to the post-hydrolysis (GDP) state. Additional structure determination of Taxol-bound microtubules at [~]2.2 [A] showed that Taxol binding converts the microtubule lattice into a pre-hydrolysis state by reversing the structural switches flipped during GTP hydrolysis. Focusing our analysis on the microtubule seam shows that the pre-hydrolysis conformation of GMPCPP or Taxol-GDP exhibits favorable lateral interactions at the seam, with lattice deformations clearly visible at the GDP seam. Together, our data show the existence of structural switches in tubulin that are coupled to the nucleotide state and are exploited by Taxol to stabilize microtubules into a pre-hydrolysis-like state. (191 words)

molecular biology↗

bioRxiv2026

Tubulin C-terminal tails are pH sensors that regulate microtubule function

Changes in intracellular pH are critical for maintaining homeostasis, mediating signaling pathways, and enabling cellular responses to stress, injury, and disease. There is increasing evidence that clusters of acidic residues, primarily glutamates, are both highly prevalent and conserved in disordered regions of proteins and can play an important role in cellular pH response. Tubulin C-terminal tails (CTTs) are glutamate rich regions which protrude from the microtubule surface. These tails are a primary site of for both post-translational modifications and binding of microtubule-associated proteins. Motivated by these observations, we measured the pH response of tubulin CTTs using NMR spectroscopy, circular dichroism, and computational simulations. We find that glutamate residues in CTTs taken from organisms across eukaryotes exhibit a robust upshift in their pKa values, that the sequential context of glutamate residues creates hot spots for protonation, and that hydrogen bonding between side chains stabilizes interactions that alter the conformation of the CTT. To determine whether the CTT pH response plays a potentially important role in microtubule interactions, we measured the pH dependence of the binding of the yeast kinesin-5, Cin8, to microtubules. We find that Cin8 binding is modulated by pH in a CTT-dependent manner. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish that pH can regulate interactions at the microtubule surface. Significance StatementVariation in cellular pH is important for cell function in changing environmental conditions or developmental states. Here we probe protonation of the glutamate-rich C-terminal tails of tubulin, revealing the existence of and mechanism driving the anomalously high pH response and subsequent regulation of microtubule binding. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish pH-based regulation of interactions at the microtubule surface.

biophysics↗

bioRxiv2025

Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

Microtubules are cytoskeletal filaments that self-assemble from the protein tubulin, a heterodimer of -tubulin and {beta}-tubulin, and are important for cell mechanics, migration, and division. Much work has focused on how the nucleotide state of {beta}-tubulin regulates the structure and dynamics of microtubules. In contrast, less is known about the structure and function of the C-terminal tails (CTTs) of - and {beta}-tubulin which are thought to freely protrude from the surface of the microtubule. To study the CTT of -tubulin, we developed three different biosensors that bind the tyrosinated -tubulin CTT (Y-CTT). Surprisingly, live imaging of the probes indicates that the Y-CTT is minimally accessible along the microtubule lattice under normal cellular conditions. Lattice binding of the Y-CTT probes can be increased by three different ways of changing the tubulin conformational state: the drug Taxol, expression of microtubule-associated proteins (MAPs) that recognize or promote an expanded tubulin conformation, or expression of tubulin that cannot hydrolyze GTP. Molecular dynamics simulations indicate that the Y-CTT undergoes numerous transient interactions with the bodies of -tubulin and {beta}-tubulin in the lattice, and that the frequency of these interactions is regulated by the tubulin nucleotide state. These findings suggest that accessibility of the Y-CTT is locally governed by nucleotide- and MAP-dependent conformational changes to tubulin subunits within the microtubule lattice.

cell biology↗

bioRxiv2025

Conformational flexibility of tubulin dimers regulates the transitions of microtubule dynamic instability

Microtubules are highly conserved polymers of {beta}-tubulin dimers that undergo dynamic instability. While dynamic instability is conserved across eukaryotes, many of its associated conformational changes, like lattice compaction and twist, are not. Tubulin dimers sample multiple conformations in solution and undergo conformational changes during polymerization; the rate and extent of these changes describes their "conformational flexibility." Here, we investigate the relationship between the conformational flexibility of tubulins and the dynamic phenotypes of the microtubules they produce with a comparative study of Drosophila melanogaster tubulin (Dm-Tb) and Bos taurus brain tubulin (Bt-Tb). While these tubulins share high sequence and structural similarity, their microtubules display divergent dynamic phenotypes in vitro, with altered transition frequencies between phases of growth and shrinkage. Dm-Tb microtubules showed drastically longer lifetimes, lower barriers to nucleation, and a high rescue frequency, while maintaining a similar growth rate to Bt-Tb microtubules. 3D reconstruction of mature Dm-Tb microtubules showed high structural conservation with mammalian microtubules. However, when we performed molecular dynamics simulations of free tubulin dimers, we found Dm-Tb to be more rigid and adopt fewer conformational states than Bt-Tb. Biochemical characterizations experimentally confirmed this finding, leading us to hypothesize that differences in the conformational flexibility of tubulins may tune the frequency of transitions between the dynamic phases of microtubules, thereby altering their stability and overall dynamic phenotypes.

biophysics↗

bioRxiv2025

Membrane-associated polymerases deliver most of the actin subunits to a lamellipodial network

Actin filaments are two-stranded protein polymers that form the basic structural unit of the eukaryotic actin cytoskeleton. While filaments assembled from purified actin in vitro elongate when soluble monomers bind to free filament ends, in cells the mechanism of filament elongation is less clear. Most monomeric actin in the cytoplasm is bound to the accessory protein profilin, and many regulators of filament assembly recruit actin-profilin complexes to membrane surfaces where they locally accelerate filament elongation. Employing quantitative live-cell imaging of actin-profilin fusion proteins and biochemically defined mutants of the branched actin regulator, WAVE1, we find that only [~]25% of the actin in leading-edge lamellipodial networks enters directly from solution, while the majority enters via membrane-associated polymerases.

cell biology↗

bioRxiv2024

Motor Clustering Enhances Kinesin-driven Vesicle Transport

Intracellular vesicles are typically transported by a small number of kinesin and dynein motors. However, the slow microtubule binding rate of kinesin-1 observed in in vitro biophysical studies suggests that long-range transport may require a high number of motors. To address the discrepancy in motor requirements between in vivo and in vitro studies, we reconstituted motility of 120-nm-diameter liposomes driven by multiple GFP-labeled kinesin-1 motors. Consistent with predictions based on previous binding rate measurements, we found that long-distance transport requires a high number of kinesin-1 motors. We hypothesized that this discrepancy from in vivo observations may arise from differences in motor organization and tested whether motor clustering can enhance transport efficiency using a DNA scaffold. Clustering just three motors improved liposome travel distances across a wide range of motor numbers. Our findings demonstrate that, independent of motor number, the arrangement of motors on a vesicle regulates transport distance, suggesting that differences in motor organization may explain the disparity between in vivo and in vitro motor requirements for long-range transport. Significance StatementIntracellular vesicles frequently travel long distances, despite having few kinesin and dynein motors. By reconstituting liposome motility with kinesin-1 motors, we demonstrate the need for high motor copy numbers for long-range transport when motors are randomly distributed on the liposome surface. We further show that motor clustering reduces the required motor number, emphasizing its potential role in enhancing transport efficiency. Our findings highlight the significance of motor organization in regulating intracellular transport and suggest that motor clustering, such as by scaffolding proteins or lipid domains, influences bidirectional transport outcomes.

biophysics↗

bioRxiv2023

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Forster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

biochemistry↗

bioRxiv2021

Kinesin-binding protein remodels the kinesin motor to prevent microtubule-binding

Kinesins are tightly regulated in space and time to control their activation in the absence of cargo-binding. Kinesin-binding protein (KIFBP) was recently discovered to bind the catalytic motor heads of 8 of the 45 known kinesin superfamily members and inhibit binding to microtubules. In humans, mutation of KIFBP gives rise to Goldberg-Shprintzen syndrome (GOSHS), but the kinesin(s) that is misregulated to produce clinical features of the disease is not known. Understanding the structural mechanism by which KIFBP selects its kinesin binding partners will be key to unlocking this knowledge. Using a combination of cryo-electron microscopy and crosslinking mass spectrometry, we determined structures of KIFBP alone and in complex with two mitotic kinesins, revealing regions of KIFBP that participate in complex formation. KIFBP adopts an alpha-helical solenoid structure composed of TPR repeats. We find that KIFBP uses a 2-pronged mechanism to remodel kinesin motors and block microtubule-binding. First, KIFBP engages the microtubule-binding interface and sterically blocks interaction with microtubules. Second, KIFBP induces allosteric conformational changes to the kinesin motor head that displace a key structural element in the kinesin motor head (-helix 4) required for microtubule binding. We identified two regions of KIFBP necessary for in vitro kinesin-binding as well as cellular regulation during mitosis. Taken together, this work establishes the mechanism of kinesin inhibition by KIFBP and provides the first example of motor domain remodeling as a means to abrogate kinesin activity.

cell biology↗

bioRxiv2020

Initial ciliary assembly in Chlamydomonas requires Arp2/3-dependent recruitment from a ciliary protein reservoir in the plasma membrane

Ciliary assembly, trafficking, and regulation are dependent on microtubules, but the mechanisms of ciliary assembly also require the actin cytoskeleton. Here, we dissect subcellular roles of actin in ciliogenesis by focusing on actin networks nucleated by the Arp2/3 complex in the powerful ciliary model, Chlamydomonas. We find the Arp2/3 complex is required for the initial stages of ciliary assembly when protein and membrane are in high demand but cannot yet be supplied from the Golgi complex. We provide evidence for Arp2/3 complex-dependent endocytosis of ciliary proteins, an increase in endocytic activity upon induction of ciliary growth, and relocalization of plasma membrane proteins to newly formed cilia. Our data support a new model of ciliary protein and membrane trafficking during early ciliogenesis whereby proteins previously targeted to the plasma membrane are reclaimed by Arp2/3 complex-dependent endocytosis for initial ciliary assembly. SUMMARYUsing the ciliary model system Chlamydomonas, we find Arp2/3 complex-mediated endocytosis is needed to reclaim cell body plasma membrane for early ciliary assembly.

cell biology↗

bioRxiv2020

Pathogenic Mutations in the Kinesin-3 Motor KIF1A Diminish Force Generation and Movement Through Allosteric Mechanisms

The kinesin-3 motor KIF1A functions in neurons where its fast and superprocessive motility is thought to be critical for long-distance transport. However, little is known about the force-generating properties of kinesin-3 motors. Using optical tweezers, we demonstrate that KIF1A and its C. elegans homolog UNC-104 undergo force-dependent detachments at ~3 pN and then rapidly reattach to the microtubule to resume motion, resulting in a sawtooth pattern of clustered force generation events that is unique among the kinesin superfamily. Whereas UNC-104 motors stall before detaching, KIF1A motors do not. To examine the mechanism of KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders, V8M and Y89D, based on their location in structural elements required for force generation in kinesin-1. Molecular dynamics simulations predict that the V8M and Y89D mutations impair docking of the N-terminal ({beta}9) or C-terminal ({beta}10) portions of the neck linker, respectively, to the KIF1A motor domain. Indeed, both mutations dramatically impair force generation of KIF1A but not the motors ability to rapidly reattach to the microtubule track. Homodimeric and heterodimeric mutant motors also display decreased velocities, run lengths, and landing rates and homodimeric Y89D motors exhibit a higher frequency of non-productive, diffusive events along the microtubule. In cells, cargo transport by the mutant motors is delayed. Our work demonstrates the importance of the neck linker in the force generation of kinesin-3 motors and advances our understanding of how mutations in the kinesin motor domain can manifest in disease.

biophysics↗

bioRxiv2017

Cytokine receptor-Eb1 interaction couples cell polarity and fate during asymmetric cell division

Asymmetric stem cell division is a critical mechanism for balancing self-renewal and differentiation. Adult stem cells often orient their mitotic spindle to place one daughter inside the niche and the other outside of it to achieve asymmetric division. It remains unknown whether and how the niche may direct division orientation. Here we discover a novel and evolutionary conserved mechanism that couples cell polarity to cell fate. We show that the cytokine receptor homolog Dome, acting downstream of the niche-derived ligand Upd, directly binds to the microtubule-binding protein Eb1 to regulate spindle orientation in Drosophila male germline stem cells (GSCs). Domes role in spindle orientation is entirely separable from its known function in self-renewal mediated by the JAK-STAT pathway. We propose that integration of two functions (cell polarity and fate) in a single receptor is a key mechanism to ensure an asymmetric outcome following cell division.

cell biology↗

bioRxiv2017

14-3-3 Negatively Regulates Actin Filament Formation In The Deep Branching Eukaryote Giardia lamblia

The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin, this function has been previously attributed to sequestration of phosphorylated cofilin. The deep branching eukaryote Giardia lamblia lacks cofilin and all other canonical actin-binding proteins (ABPs), and 14-3-3 was identified as an actin-associated protein in Giardia, yet its role in actin regulation was unknown. Gl14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia's sole Rho family GTPase, GlRac, modulates Gl14-3-3's association with actin, providing the first connection between GlRac and the actin cytoskeleton in Giardia. Giardia actin contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a negative regulator of actin filament formation.\n\nImportanceGiardia lacks canonical actin binding proteins. 14-3-3 was identified as an actin interactor but the significance of this interaction was unknown. Loss of 14-3-3 results in ectopic short actin filaments, indicating that 14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that 14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardias sole Rho family GTPase, GlRac, this result provides the first mechanistic connection between GlRac and actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between 14-3-3 and actin suggesting that 14-3-3 is regulating multiple actin complexes. Overall, we find that 14-3-3 is a negative regulator of actin filament formation in Giardia.

cell biology↗