Biology subjects

Akhmanova, A.

Publications and source records attributed to Akhmanova, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic proteomics identifies a conserved mechanism for polar targeting of plant cortical proteins

Multicellular development is tightly coupled to the polarization of individual cells, which partitions polar proteins along the cell cortex and can control asymmetric cell division, anisotropic growth, local differentiation or physiology. Mechanisms driving cell polarization have been described in fungi and animals, but these lack counterparts in plants. While several polarized proteins have been identified in plants, the overall mechanisms guiding their polar localization are poorly characterized. Through iterative affinity proteomics on the recently identified SOSEKI polar proteins, we discovered a network of polar proteins that is conserved in the flowering plant Arabidopsis and the liverwort Marchantia. We next used this collection of novel polarized proteins for systematic proximity ligation proteomics in these two species to map their global polar proteome. We identified a subfamily of polarized Protein S-acyl transferase (PAT) enzymes that are required for membrane targeting of Arabidopsis SOSEKI proteins. Using human and fruit fly models, we showed that PAT19 is sufficient for membrane targeting of SOSEKI1, likely through direct palmitoylation. This work demonstrates a conserved mechanism for polar protein targeting in plants and offers a resource for studying polar protein localization.

plant biology↗

bioRxiv2026

Dissection of centrosomal γ-TuRC activation pathways controlling microtubule density in interphase cells

Animal microtubule-organizing centers, including the centrosome and the Golgi apparatus, regulate microtubule nucleation and anchoring through the {gamma}-tubulin ring complex ({gamma}-TuRC) and CAMSAP-mediated minus-end stabilization. However, functional redundancy between these pathways has impeded dissection of their contributions to controlling microtubule organization and density. Here, we addressed this problem using combinatorial gene knockouts, protein depletions and Expansion Microscopy. By simultaneously eliminating CAMSAP2 and the {gamma}-TuRC-targeting proteins AKAP450, pericentrin, CDK5RAP2, myomegalin, ninein and AKNA, we generated viable RPE1 cells that lack both Golgi-derived microtubules and {gamma}-TuRC localization within the pericentriolar material and at subdistal appendages. Despite the disruption of these major microtubule-organizing pathways, overall microtubule density was only partially reduced. The remaining microtubules depended on CEP192 and NEDD1, which, together with ch-TOG, can activate {gamma}-TuRC at the centriole wall, in acentriolar cells, and in biochemical reconstitution assays. Our results demonstrate that in the absence of CAMSAP-mediated stabilization, interphase microtubule formation strongly relies on {gamma}-TuRC activation, which occurs through several redundant pathways.

cell biology↗

bioRxiv2026

Microtubule Engagement by the WD40-Containing Tail of Kinesin-4 KIF21B

Kinesin tails are structurally diverse and mediate a range of functions, including autoinhibition, cargo binding, and microtubule regulation. Within the kinesin-4 family, KIF21A and KIF21B have emerged as key regulators of microtubule network organization and dynamics in neurons and immune cells, yet the molecular basis of this activity has remained unclear. Here, we combined single-particle cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to examine how the KIF21B tail engages microtubules. We find that conserved residues in the WD40 {beta}-propeller and an adjacent N-terminal linker contact successive tubulin dimers along a single protofilament, forming an extended longitudinal binding mode that spans both the intradimer and interdimer interfaces. Cryo-EM 3D classification further revealed two distinct engagement states, a tilted state, in which the {beta}-propeller makes partial contacts with the microtubule while the linker remains anchored, and a flat state, in which the {beta}-propeller lies flush with the lattice surface. Cryo-ET of full-length KIF21B reveals multiple binding configurations on the microtubule lattice, including orientations consistent with crosslinking adjacent microtubules. Together, these findings provide a structural framework for tail-mediated microtubule attachment by a neuronal kinesin-4 and suggest how its distinctive WD40 domain may contribute to microtubule regulation and organization of microtubule arrays.

biochemistry↗

bioRxiv2026

Structure of the mammalian ciliary transition zone microtubule doublet

The ciliary transition zone gates bidirectional protein trafficking to maintain the specialized ciliary proteome using microtubule doublets as a scaffold. While ciliary axonemal doublets are well-characterized, the molecular architecture of the transition zone doublet remains elusive. Here, we report the structure of the mammalian transition zone doublet from bovine tracheal cilia using cryo-electron tomography at 4.7-5.0 [A] resolution. The transition zone doublet is a structurally independent segment defined by an 8 nm-periodic arrangement of unique microtubule-inner proteins (MIPs) and microtubule-associated proteins (MAPs). We identify the calcium-binding protein CAPSL as a lumenal MIP that forms a pseudo-helical spiral and stabilizes microtubules in vitro. Furthermore, a dense MAP network on the A-microtubule surface clashes with intraflagellar transport (IFT) motor binding sites, suggesting anterograde IFT is directed to the B-microtubule for potential regulation by MAP9. Our work provides a structural framework for understanding gated ciliary transport and transition zone-linked human ciliopathies.

Cell Biology↗

bioRxiv2026

Regulation of microtubule abundance and minus end dynamics by Katanin, CAMSAPs, WDR47 and kinesin-13

Microtubule networks are major determinants of cell architecture and logistics. Microtubule organization and density are regulated by severing enzymes, which cut microtubule lattices or affect their growth and shortening. These activities can lead to microtubule amplification or disassembly, depending on the presence of microtubule stabilizers or destabilizers, but the interplay between these factors is poorly understood. Here, we reconstituted in vitro the activity of microtubule severase katanin together with microtubule minus-end stabilizers CAMSAPs, their binding partner WDR47 and microtubule depolymerase kinesin-13/MCAK. We confirmed that katanin can amplify or destroy microtubules in a concentration-dependent manner. CAMSAPs recruit katanin to microtubules and reduce katanin concentration needed for both amplification and destruction, whereas kinesin-13 completely abolishes microtubule amplification. WDR47 binds to microtubules decorated by CAMSAPs and suppresses katanin binding and severing. In addition, both katanin and WDR47 inhibit polymerization of CAMSAP-decorated microtubule minus ends. These data explain how these proteins act together to fine-tune microtubule minus-end stability without strongly increasing microtubule abundance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/714132v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@746fe3org.highwire.dtl.DTLVardef@5dd5a8org.highwire.dtl.DTLVardef@762373org.highwire.dtl.DTLVardef@1192db_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

cell biology↗

bioRxiv2025

Taxane-Induced Conformational Changes in the Microtubule Lattice Activate GEF-H1-Dependent RhoA Signaling

Taxanes are widely used chemotherapeutic agents that perturb cell division. They also exert effects during interphase, but the underlying mechanisms are poorly understood. Here, we show that taxanes activate RhoA signaling and induce actin remodeling by displacing the RhoA activator GEF-H1 from microtubules. This taxane-induced release of GEF-H1 occurs rapidly, is independent of tubulin post-translational modifications, and can be recapitulated using purified proteins. In vitro reconstitution assays combined with analyses of microtubule structure revealed that microtubule binding by GEF-H1 is inhibited by microtubule-stabilizing agents that expand the microtubule lattice, such as taxanes and GMPCPP, but not by others, including GTP{gamma}S and discodermolide, which stabilize a compacted microtubule lattice. Our findings demonstrate that alterations in microtubule lattice conformation can activate key signaling pathways, offering new insights into the mode of action of taxanes and the possible origins of their side effects.

cell biology↗

bioRxiv2025

Rab27-dependent egress of SARS-CoV-2 via secretory amphisomes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, hijacks host cellular machinery to replicate and spread. Understanding how SARS-CoV-2 reorganizes host cell architecture to accommodate this is essential for elucidating its pathogenesis and identifying therapeutic targets. While the molecular mechanisms of SARS-CoV-2 entry are well characterized, the pathways governing viral egress remain incompletely understood. Conventional approaches such as transcriptomics and electron microscopy have provided valuable insights but lack the combined spatial and molecular resolution needed to map these processes within intact cells. Here, we apply Ten-fold Robust Expansion Microscopy (TREx) to visualize SARS-CoV-2-induced remodeling of the endolysosomal system in multiciliated cells of primary human airway epithelial tissue. This approach reveals Golgi fragmentation and the formation of enlarged virus-containing organelles. Analysis of endolysosomal markers in Vero E6 cells shows that these structures are positive for CD63, Rab7, and LC3, consistent with amphisome identity. Moreover, pharmacological inhibition of Rab27-dependent amphisome-plasma membrane fusion with Nexinhib20 reduces viral infection, implicating secretory autophagy as a pathway for SARS-CoV-2 egress. These findings establish expansion microscopy as a powerful tool for spatial virology and uncover a Rab27-mediated amphisome fusion mechanism as a druggable route for SARS-CoV-2 release.

cell biology↗

bioRxiv2025

Immortalization of human nasal and bronchial airway epithelial cells for genome editing applications

In vitro air-liquid interface culture of airway epithelial cells is used as a model system to study respiratory diseases. This culture system not only overcomes the need for animal models or continuous biopsies from individuals but also enables studies of pathophysiology associated with the disease in a patient background. Human airway basal cells serve as progenitor cells for a functional pseudostratified airway epithelium composed mainly of multiciliated and secretory cells. However, due to the limited ability of basal cells to proliferate and differentiate, the long-term use of primary material in culture is restricted. This challenges research that requires genome editing. Here, we describe airway stem cells from nasal and bronchial origin immortalized by hTERT overexpression followed by polyclonal expansion. We demonstrate that this diverse panel of cell lines shows differentiation patterns similar to primary stem cells and can be used for lentiviral and CRISPR/Cas9 genome editing. These cell lines and optimized protocols facilitate airway biology research and disease phenotyping.

cell biology↗

bioRxiv2025

Logic-gating the HaloTag system with Conditional-Halo-ligator 'CHalo' reagents

HaloTag proteins spontaneously ligate onto any chemical reagent featuring a chloroalkane motif (CA). We introduce the conditional CHalo motif, which ligates to HaloTag only after uncaging by light or enzymes. (1) Photo-triggered CHalo fluorogenic reagents allow spatiotemporally-specific labeling; (2) photo-triggered CHalo heterodimerisers can photocontrol protein recruitment; and (3) enzyme-triggered CHalo reagents can durably record diverse enzyme activities, and multiplexing them should allow quantitative ratiometric recording of multiple activities in parallel. CHalo thus permits manifold extensions to the HaloTag technology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/676741v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d18cd6org.highwire.dtl.DTLVardef@175694forg.highwire.dtl.DTLVardef@154ffd5org.highwire.dtl.DTLVardef@167fec2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Microtubule organization and molecular architecture of ciliary basal bodies in multiciliated airway cells

Microtubule organization depends on cell type and function. Microtubule networks of many differentiated cell types, such as epithelial cells, are poorly understood due to their high density. Here, we used expansion microscopy to quantitatively describe the three-dimensional organization of the microtubule network in human airway multiciliated cells. In these cells, most apical and apicobasal microtubules nucleate and anchor at the basal foot, a part of the ciliary basal body. Using a newly developed volumetric averaging tool, we generated a high-resolution 3D map of the basal body, delineating the position of structural components and proteins involved in microtubule nucleation and anchoring. {gamma}-TuRC, its binding partners NEDD1 and Augmin/HAUS, and centriolar appendage proteins ninein and AKNA localize to the basal foot. Functional analyses demonstrated that NEDD1, but not ninein or HAUS are essential for basal foot-dependent microtubule organization. Our data reveal the distinct architecture of microtubule-organizing centers responsible for the formation of dense microtubule arrays in multiciliated cells.

cell biology↗

bioRxiv2025

Weak Motifs, Strong Complex: KANK1 Uses Cooperative Multivalency with the Hub-Protein LC8 to Bridge Cytoskeletal Complexes

The cortically anchored adaptor KANK1 organizes microtubules at focal adhesions through a long, intrinsically disordered linker (L2), yet how this linker spans the [~]35-50 nm membrane-microtubule gap is unclear. Here, we combine in-cell, biochemical, and biophysical assays, predictions of motif interaction and multivalent assembly using AlphaFold, and structural analysis by electron microscopy to show that the hub protein LC8, which binds more than 100 clients, converts the intrinsically disordered 600 amino acid L2 into an elongated, multivalent, rod-like assembly. In contrast, isolated motif peptides fail to bind LC8 at physiologically relevant concentrations, indicating that strong complex formation arises from cooperativity among multiple weak sites. These results establish LC8 as a molecular switch that rigidifies and extends KANK1 L2 via distributed weak motifs and short linkers. This interaction produces compositionally homogeneous yet conformationally adaptable rods, long enough to bridge the membrane-microtubule gap, resolving the paradox. This work expands the LC8 binding repertoire, reveals design principles for multivalent assembly, and suggests a generalizable strategy for tuning length, rigidity, and flexibility in large protein architectures.

biophysics↗

bioRxiv2025

SSNA1 organizes the distal luminal centriolar network and promotes ciliogenesis without microtubule association

The distal lumen of the centriole plays a critical role in ciliogenesis, yet the molecular composition, spatial organization, and targeting hierarchy of distal luminal proteins remain poorly understood. In this study, we identify Sjogrens syndrome nuclear autoantigen 1 (SSNA1) as a bona fide centriolar protein and a key regulator of ciliogenesis in mammalian cells. Using our newly developed knockout (KO)-validated antibody, we show that, contrary to previous reports, SSNA1 does not reside in the nucleus, midbody, or ciliary axoneme. Instead, super-resolution imaging combined with expansion microscopy (ExM) reveals that SSNA1 localizes to the distal lumen of centrioles and the basal bodies of both primary and motile cilia, where it is arranged in a ring-like configuration with 9-fold symmetry and apart from centriolar microtubules. Molecular dissection using tag-free SSNA1, its oligomerization-deficient mutants, and microtubule co-pelleting assays further demonstrates that SSNA1, previously described as a microtubule nucleator, stabilizer, and branching factor, does not bind microtubules in vitro. Interactor screening and KO analysis unveil a hierarchical targeting network involving a C2CD3-SSNA1-LRRCC1 axis in the distal lumen. Functional characterization indicates that although dispensable for cell division, overall centriole organization and duplication, SSNA1 promotes cilia assembly by facilitating CP110 removal. Our findings redefine the physiological role of SSNA1 as part of the distal luminal module contributing to ciliogenesis and provide new insights into the molecular architecture and functional relevance of the distal centriolar lumen. SignificanceCentrioles template the assembly of cilia and flagella, whose dysfunction causes human diseases known as ciliopathies. Key centriolar structures - the cartwheel in the proximal lumen, the inner scaffold in the central lumen, and the distal and subdistal appendages outside the distal lumen - have been extensively characterized. However, the molecular organization and functional relevance of the distal lumen remain largely unexplored. Here, we identify SSNA1 as a novel centriolar component at the distal lumen and an essential regulator of ciliogenesis. Our work challenges the long-held view of SSNA1 as a microtubule-associated protein and provides new insights into the architecture, interaction network, targeting hierarchy, and function of the distal centriolar lumen, exemplifying how it modulates centriolar organization and drives ciliogenesis.

cell biology↗

bioRxiv2024

Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but reduced cell stiffness. Mitochondria and endoplasmic reticulum sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.

cell biology↗

bioRxiv2024

A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

Cilia are essential motile or sensory organelles found on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, however the underlying biochemical mechanisms are largely unexplored. Here, we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins, CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules, and, when mutated, cause ciliopathies such as Joubert syndrome. CEP104, a protein containing a tubulin-binding TOG domain, is an inhibitor of microtubule growth and shortening that interacts with EBs on the microtubule surface and with a luminal microtubule-pausing factor CSPP1. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics directly but act as scaffolds for their partners. Cryo-electron tomography showed that together, ciliary tip module members form plus-end-specific cork-like structures which reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.

cell biology↗

bioRxiv2022

GelMap: Intrinsic calibration and deformation mapping for expansion microscopy

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy by physically expanding biological specimen in three dimensions. Nonetheless, using ExM for quantitative or diagnostic applications requires robust quality control methods to precisely determine expansion factors and to map deformations due to anisotropic expansion. Here we present GelMap, a flexible workflow to introduce a fluorescent grid into pre-expanded hydrogels that scales with expansion and reports deformations. We demonstrate that GelMap can be used to precisely determine the local expansion factor and to correct for deformations without the use of cellular reference structures or pre-expansion ground truth images. Moreover, we show that GelMap aids sample navigation for correlative uses of expansion microscopy. Finally, we show that GelMap is compatible with expansion of tissue and can be readily implemented as a quality control step into existing ExM workflows.

cell biology↗

bioRxiv2022

CAMSAP-driven microtubule release from γ-TuRC and its regulation by nucleation-promoting factors

{gamma}-tubulin ring complex ({gamma}-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from {gamma}-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here, we reconstituted this process in vitro using purified components. We found that all CAMSAP proteins could bind to the minus-ends of {gamma}-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends, but not the minus-end tracking protein CAMSAP1 induced microtubule release from {gamma}-TuRC. CDK5RAP2, a {gamma}-TuRC-interactor, and CLASP2, a regulator of microtubule growth, stimulated {gamma}-TuRC-dependent microtubule nucleation, but only CDK5RAP2 inhibited CAMSAP-driven microtubule detachment by suppressing CAMSAP binding to {gamma}-TuRC-anchored minus ends. CDK5RAP2 also improved {gamma}-TuRC selectivity for 13-rather than 14-protofilament microtubules in microtubule-capping assays. Our results support a model whereby CAMSAPs exploit an imperfect attachment between {gamma}-TuRC and the nucleated microtubule to promote minus-end elongation and release, whereas CDK5RAP2 improves the fit between {gamma}-TuRC and 13-protofilament microtubules and enhances nucleation.

cell biology↗

bioRxiv2022

CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates elongation and stability of ciliary axonemes and other microtubule-based structures.

cell biology↗

bioRxiv2022

Estimation of microtubule-generated forces using a DNA origami nanospring

Microtubules are dynamic cytoskeletal filaments that can generate forces when polymerizing and depolymerizing. Proteins that follow growing or shortening microtubule ends and couple forces to cargo movement are important for a wide range of cellular processes. Quantifying these forces and the composition of protein complexes at dynamic microtubule ends is challenging and requires sophisticated instrumentation. Here we present an experimental approach to estimate microtubule-generated forces through the extension of a fluorescent spring-shaped DNA origami molecule. Optical readout of the spring extension enables recording of force production simultaneously with single-molecule fluorescence of proteins getting recruited to the site of force generation. DNA nanosprings enable multiplexing of force measurements and only require a fluorescence microscope and basic laboratory equipment. We validate the performance of DNA nanosprings against results obtained using optical trapping. Finally, we demonstrate the use of the nanospring to study proteins that couple microtubule growth and shortening to force generation.

biophysics↗