Microtubule Structure Research Articles

Modeling and Predicting Second-Harmonic Generation from Protein Molecular Structure

Polarization resolved second-harmonic generation (pSHG) microscopy is increasingly used for mapping organized arrays of noncentrosymmetric proteins such as collagen, myosin, and tubulin, and holds potential for probing their molecular structure and supramolecular organization in intact tissues. However, the contrast mechanism of pSHG is complex and the development of applications in the life sciences is hampered by the lack of models accurately relating the observed pSHG signals to the underlying molecular and macromolecular organization. In this work, we establish a general multiscale numerical framework relating the micrometer-scale SHG measurements to the atomic-scale and molecular structure of the proteins under study and their supramolecular arrangement. We first develop a new method to automatically analyze pSHG signals independently of the protein type and fiber orientation. We then characterize experimentally pSHG signals in live zebrafish larvae and show that they can be used to distinguish collagen, myosin, and tubulin structures in intact tissues. We then introduce a numerical model that considers the peptide bond (PB) as the elementary SHG source in proteins and takes into account the three-dimensional (3D) distribution of PBs to predict the second-order hyperpolarizability tensor β of proteins, as well as the SHG efficiency and pSHG response of an arbitrary macromolecular assembly. We show that this model accurately reproduces pSHG measurements obtained from collagen, myosin, microtubule, and actin structures, revealing the precise dependence of SHG signals on the 3D distribution of PBs within protein assemblies. We then use our model to analyze pSHG from a 3D distribution of microtubule assemblies as a function of out-of-plane angles, angular disorder, and polarity. Finally, we demonstrate that our model predicts SHG from different molecular conformations of tubulin that are highly relevant from a biomedical point of view as associated with microtubules (de)polymerization. By bridging scales from the molecular bonds to the optical wavelength, our model provides an accurate interpretation of SHG signals in terms of protein structure and supramolecular organization. Published by the American Physical Society 2024

Synthesis, biological and computational evaluation of novel cyanomethyl vinyl ether derivatives.

This work explores the biological evaluation of novel cyanomethyl vinyl ether derivatives as antiproliferative agents. Tubulin, crucial to microtubule structure and function, is a target for cancer therapies. In vitro cytotoxicity assessments revealed significant activity in SKOV3 ovarian carcinoma cells and A549 lung carcinoma cells. Structure-Activity Relationship (SAR) analysis indicated that the E isomer and specific substitutions influenced the biological activity. Computational assays predicted favorable ADME properties, highlighting potential as anticancerous agents. Molecular docking studies demonstrated that compound 12E, with the E geometry of the double bond and fused polyaromatic rings such as phenanthrene, has robust interaction with tubulin, suggesting enhanced stability due to diverse amino acid interactions. Comparative spatial distributions with colchicine further indicated potential mechanistic similarities.

Sexually dimorphic dynamics of the microtubule network in medaka (Oryzias latipes) germ cells.

Gametogenesis is the process through which germ cells differentiate into sexually dimorphic gametes, eggs and sperm. In the teleost fish medaka (Oryzias latipes), a germ cell-intrinsic sex determinant, foxl3, triggers germline feminization by activating two genetic pathways that regulate folliculogenesis and meiosis. Here, we identified a pathway involving a dome-shaped microtubule structure that may be the basis of oocyte polarity. This structure was first established in primordial germ cells in both sexes, but was maintained only during oogenesis and was destabilized in differentiating spermatogonia under the influence of Sertoli cells expressing dmrt1. Although foxl3 was dispensable for this pathway, dazl was involved in the persistence of the microtubule dome at the time of gonocyte development. In addition, disruption of the microtubule dome caused dispersal of bucky ball RNA, suggesting the structure may be prerequisite for the Balbiani body. Collectively, the present findings provide mechanistic insight into the establishment of sex-specific polarity through the formation of a microtubule structure in germ cells, as well as clarifying the genetic pathways implementing oocyte-specific characteristics.

Monodispersed and Monofunctionalized DNA-Caged Au Nano-Clusters with Enhanced Optical Properties for STED Imaging.

The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugatesonly achieved ≈55nm resolution and yielded spotted,poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging.

Design, Synthesis, and Evaluation of An Anti-trypanosomal [1,2,4]Triazolo[1,5-a]pyrimidine Probe for Photoaffinity Labeling Studies.

Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

The effects of chronic morphine administration on spatial memory and microtubule dynamicity in male mice's brain

The examination of the influence of morphine on behavioral processes, specifically learning and memory, holds significant importance. Additionally, microtubule proteins play a pivotal role in cellular functions, and the dynamics of microtubules contribute to neural network connectivity, information processing, and memory storage. however, the molecular mechanism of morphine on microtubule dynamics, learning, and memory remains uncovered. In the present study, we examined the effects of chronic morphine administration on memory formation impairment and the kinetic alterations in microtubule proteins induced by morphine in mice. Chronic morphine administration at doses of 5 and 10 mg/kg dose-dependently decreased subjects' performance in spatial memory tasks, such as the Morris Water Maze and Y-maze spontaneous alternation behavior. Furthermore, morphine was found to stabilize microtubule structure, and increase polymerization, and total polymer mass. However, it simultaneously impaired microtubule dynamicity, stemming from structural changes in tubulin dimer structure. These findings emphasize the need for careful consideration of different doses when using morphine, urging a more cautious approach in the administration of this opioid medication.

Transition of human γ-tubulin ring complex into a closed conformation during microtubule nucleation.

Microtubules are essential for intracellular organization and chromosome segregation. They are nucleated by the γ-tubulin ring complex (γTuRC). However, isolated vertebrate γTuRC adopts an open conformation that deviates from the microtubule structure, raising the question of the nucleation mechanism. In this study, we determined cryo-electron microscopy structures of human γTuRC bound to a nascent microtubule. Structural changes of the complex into a closed conformation ensure that γTuRC templates the 13-protofilament microtubules that exist in human cells. Closure is mediated by a latch that interacts with incorporating tubulin, making it part of the closing mechanism. Further rearrangements involve all γTuRC subunits and the removal of the actin-containing luminal bridge. Our proposed mechanism of microtubule nucleation by human γTuRC relies on large-scale structural changes that are likely the target of regulation in cells.

Microtubule polyglutamylation is an essential regulator of cytoskeletal integrity in Trypanosoma brucei.

Tubulin polyglutamylation, catalysed by members of the tubulin tyrosine ligase-like (TTLL) protein family, is an evolutionarily highly conserved mechanism involved in the regulation of microtubule dynamics and function in eukaryotes. In the protozoan parasite Trypanosoma brucei, the microtubule cytoskeleton is essential for cell motility and maintaining cell shape. In a previous study, we showed that T. brucei TTLL6A and TTLL12B are required to regulate microtubule dynamics at the posterior cell pole. Here, using gene deletion, we show that the polyglutamylase TTLL1 is essential for the integrity of the highly organised microtubule structure at the cell pole, with a phenotype distinct from that observed in TTLL6A- and TTLL12B-depleted cells. Reduced polyglutamylation in TTLL1-deficient cells also leads to increased levels in tubulin tyrosination, providing new evidence for an interplay between the tubulin tyrosination and detyrosination cycle and polyglutamylation. We also show that TTLL1 acts differentially on specific microtubule doublets of the flagellar axoneme, although the absence of TTLL1 appears to have no measurable effect on cell motility.

Chromosomal passenger complex condensates generate parallel microtubule bundles in vitro

The mitotic spindle contains many bundles of microtubules (MT) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the Chromosomal Passenger Complex (CPC) purified from E. coli undergoes liquid-liquid demixing (LLD) in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, we found the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLD or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce microtubule bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain microtubule organizing centers that generate microtubule bundles that emerge with the opposite polarity from centrosomes.

Polyaniline-coated kapok fibers for convenient in-syringe solid-phase microextraction and determination of organochlorine and pyrethroid pesticide residues in aqueous samples

Pesticides used in agriculture have low polarity and a tendency to accumulate in fatty tissues, posing potential risks to human health. Effective pre-treatment is crucial due to complex sample matrices and low concentrations of pesticide residues typically encountered in instrument analysis. In this study, polyaniline-coated kapok fiber (PANI-KF) was synthesized successfully using in-situ oxidative polymerization for use as sorbents in in-syringe SPME of pyrethroid pesticides (PYRs) and organochlorine pesticides (OCPs) from aqueous samples. Coating the natural KF with PANI maintained the hollow microtubular structure and fiber morphology while significantly enhancing the extraction efficiency. The extraction process was easily conducted by simply pulling and pushing the syringe plunger. The entire extraction process, utilizing 3 mg of PANI-KF, could be completed in approximately 3 min. Density functional theory results indicated that the adsorption mechanism of PANI-KF towards OCPs and PYRs mainly involved van der Waals interactions, π-π interactions, and weak hydrogen bonding interactions. With the coupling of gas chromatography-mass spectrometry, a quantification method was established that exhibited good linearities (R2 > 0.990), and relative recoveries (87.2–108.5 %). The limits of detection ranged from 0.4 to 2.0 ng mL−1 and the matrix effects were negligible (−12.3–16.4 %). The validated in-syringe SPME-GC-MS method was successfully applied to determine pesticide residues in fruit juices, oral liquids and herbal extract granules with satisfactory accuracy and precision. PANI-KF exhibits remarkable promise as a sorbent for the extraction and enrichment of pesticide residues in aqueous samples, thereby contributing to the advancement of pesticide residue determination methodologies.

Centriole and transition zone structures in photoreceptor cilia revealed by cryo-electron tomography.

Primary cilia mediate sensory signaling in multiple organisms and cell types but have structures adapted for specific roles. Structural defects in them lead to devastating diseases known as ciliopathies in humans. Key to their functions are structures at their base: the basal body, the transition zone, the "Y-shaped links," and the "ciliary necklace." We have used cryo-electron tomography with subtomogram averaging and conventional transmission electron microscopy to elucidate the structures associated with the basal region of the "connecting cilia" of rod outer segments in mouse retina. The longitudinal variations in microtubule (MT) structures and the lumenal scaffold complexes connecting them have been determined, as well as membrane-associated transition zone structures: Y-shaped links connecting MT to the membrane, and ciliary beads connected to them that protrude from the cell surface and form a necklace-like structure. These results represent a clearer structural scaffold onto which molecules identified by genetics, proteomics, and superresolution fluorescence can be placed in our emerging model of photoreceptor sensory cilia.

Catkin-derived carbon microtubes for hybrid anode materials of high-performance lithium ion batteries

As a common biological waste, catkin shows negligible economic value and is hazardous to the environment and human respiratory health. To turn waste into treasure, in this work, a hybrid anode is successfully prepared by using the catkin-derived 1D hollow carbon microtube as dispersing agents to well disperse the MnO particles via a simple one-step pyrolysis method. Benefiting from the high electrical conductivity and hollow microtubule structure of the catkin-derived carbon, as well as the evenly dispersion of the MnO particles and the interfacial energy storage of Mn0/Li2O interfaces, the lithium ion battery based on the resultant MnO/C hybrid anode exhibits outstanding rate performance (high reversible specific capacity of 1365 mAh g−1 at a current density of 0.1 A g−1 and maintains 451 mAh g−1 even at a high current density of 6.4 A g−1) and superior cycling stability (retains ∼100 % after 1000 cycles at a current density of 0.8 A g−1). This work conforms to the development concept of green environmental protection and realizes the unity of economic and social benefits.

Cellulose/silica composite microtubular superfoam with excellent flame retardancy, thermal insulation and ablative resistance.

Thermal insulation materials with good flame-retardant properties have attracted widespread attention because of their huge application potential. Traditional petrochemical-based polymer insulation materials are flammable and have problems with environmental pollution. The microtubule structure is a perfect microstructure with excellent thermal insulation performance. In addition, the microtubule structure also has low density and high elasticity. Therefore, the microtubule structure is an important reference microstructure for the development of efficient thermal insulation materials. In this paper, a cellulose/SiO2 composite microtube thermal insulation superfoam has been successfully prepared. Cellulose microtubules were successfully prepared from poplar sawdust by chemical methods. The SiO2 aerogel precursor solution can be quickly adsorbed by the delignified cellulose microtubes. The SiO2 aerogel shells are evenly distributed only on the inner and outer walls of the delignified cellulose microtubes. The cellulose/SiO2 microtube composite (CSMC) superfoam exhibits low density, good mechanical properties, and low thermal conductivity (as low as 0.042 ± 0.0018 W m-1 K-1). The CSMC superfoam exhibits excellent self-extinguishing and flame-retardant properties. After being burned by a butane flame, the superfoam still has certain mechanical properties. The thermal conductivity of the B-CSMC superfoam (the CSMC superfoam burned by a butane flame) is about 0.050 W m-1 K-1. The B-CSMC superfoam remained almost unchanged after being continuously ablated by a butane flame for 3600 seconds.

Carbon/ZrO2 aerogel composite microtube superfoam.

High-performance thermal insulation materials with broad application prospects have attracted great attention. The introduction of new microstructures into thermal protection materials can significantly improve the thermal insulation performance. The tubular microstructure has obvious advantages such as thermal insulation, lightweight, mechanical, and other properties. Therefore, the microtubular structure has become an important reference microstructure for the development of high-performance thermal insulation materials. In this paper, the carbon/ZrO2 aerogel composite microtube superfoams with excellent thermal protection properties were prepared by a vacuum filtration and high-temperature carbonization method. The ZrO2 aerogel precursor solution can be quickly and uniformly adsorbed on the inner and outer walls of cellulose microtubules. These adsorbed ZrO2 aerogel precursor solution films can be converted into ZrO2 alcohol gel shells under the acceleration and promotion effect of citric acid at 65 °C. The micromorphology of the ZrO2 aerogel shell on the inner and outer walls of the composite microtubes can be efficiently controlled by the concentration of the ZrO2 aerogel precursor solution and the carbonization temperature. The carbon/ZrO2 aerogel composite microtube superfoam exhibits a lower thermal conductivity, lower density, good mechanical properties, and high ablation resistance. The thermal conductivity of the carbon/ZrO2 aerogel composite microtube superfoam is as low as 0.040 ± 0.001 W m-1 K-1. The residual rate of the carbon/ZrO2 aerogel composite microtube superfoam is still as high as 84.33% after butane flame ablation for up to 3600 seconds.

Thermal control materials of carbon/SiO2 composites with a honeycomb structure.

Anisotropic composite thermal control materials show efficient thermal management ability, which can not only improve the heat flow in the direction of high thermal conductivity and prevent local overheating, but also reduce the heat flow in the direction of the low thermal conductivity and improve thermal insulation. In this paper, the anisotropic microstructure of natural wood (e.g. poplar) was used as a reference template. The filling of the SiO2 aerogel into the multi-layer pore structure and microtubule structure originally occupied by lignin was controlled by the process of the axial self-adsorption, limited sol-gel and natural drying. The anisotropic composite biomimetic thermal control materials were prepared by high temperature carbonization and the thermal control properties were evaluated. The anisotropic carbon/SiO2 (ACS) composite biomimetic thermal control material obtained after carbonization inherits the anisotropic microstructure. The axial thermal conductivity of the ACS composite biomimetic thermal control material is mainly determined by the thermal conductivity of the carbon composite microtubule structure. The SiO2 aerogel filled between carbon composite microtubules and the abundant axial microcracks endow the ACS composite biomimetic thermal control material with excellent radial thermal insulation performance. The maximum specific surface area of the ACS composite biomimetic thermal control material is 183 m2 g-1, and the maximum density is 517.8 ± 55.9 kg m-3. The maximum axial thermal conductivity of the ACS composite biomimetic thermal control material is 0.73 ± 0.07 W m-1 k-1, and the radial thermal conductivity is 0.28 ± 0.01 W m-1 k-1. The maximum ratio of the axial thermal conductivity to radial thermal conductivity of the ACS composite biomimetic thermal control material is 3.3.

Organization of two kinesins in a two-dimensional microtubule network.

In intracellular active transport, molecular motors are responsible for moving biological cargo along networks of microtubules that serve as scaffolds. Cargo dynamics can be modified by different features of microtubule networks such as geometry, density, orientation modifications. Also, the dynamical behaviour of the molecular motors is determined by the microtubule network and by the individual and/or collective action of the motors. For example, unlike single kinesins, the mechanistic behavior of multiple kinesins varies from one experiment to another. However, the reasons for this experimental variability are unknown. Here we show theoretically how non-radial and quasi-radial microtubule architectures modify the collective behavior of two kinesins attached on a cargo. We found out under which structural conditions transport is most efficient and the most likely way in which kinesins are organized in active transport. In addition, with motor activity, mean intermotor distance and motor organization, we determined the character of the collective interaction of the kinesins during transport. Our results demonstrate that two-dimensional microtubule structures promote branching due to crossovers that alter directionality in cargo movement and may provide insight into the collective organization of the motors. Our article offers a perspective to analyze how the two-dimensional network can modify the cargo-motor dynamics for the case in which multiple motors move in different directions as in the case of kinesin and dynein.

Cationic human defence peptides and cornea HSV‐1 infections: Results of a rabbit study

In this study, we evaluated the potential of LiQD cornea glue supplemented with LL37‐derived peptide GF19, delivered through Silicium nanoparticles (SiNP) to prevent herpes simplex virus type 1 (HSV‐1) infection in a rabbit corneal model. SiNP‐GF19 was synthesized using a hydrophobic core, a hydrophilic shell, and a reactive surface that allowed covalent bonding to the collagen‐like peptides of our developed LiQD Cornea glue. Our results showed that SiNP‐GF19 was able to effectively release GF19 in a sustained manner, with approximately 60% of the peptide being released over 24 h. The remaining 40% of GF19 is trapped in the SiNP and can only be released upon complete degradation of the nanoparticles, which may occur via cellular uptake and enzymatic degradation. This ensures the prolonged release of GF19. The mechanism of action of GF19 against HSV‐1 may involve preventing the binding of the virus to host cells. Previous studies have shown that LL37, from which GF19 is derived, can block HSV‐1 binding to host cells, leading to decreased viral infection. However, in our study, we did not see reduced viral shedding, furthermore, nanoparticles coated with the GF19 peptide or nanoparticles themselves may have an eye irritant effect. Nevertheless, LiQD Cornea supplemented with SiNP‐GF19 or LiQD alone were effective in promoting corneal regeneration and healing. Specifically, it led to the formation of a relatively mature epithelium composed of 2–3 epithelial layers, expressing CK3 in the outermost epithelial layer, compared to the absence of epithelium or a monolayer of unstructured CK3‐negative epithelium in the cyanoacrylate adhesive control group. LiQD Cornea formulation without SiNP‐GF19 also displayed significantly lower α‐SMA staining than the rest of the experimental groups in the implant area, which overexpression is associated with abnormal extracellular matrix deposition and scar formation. In terms of nerve regeneration, all LiQD Cornea formulations showed βIII tubulin expression, indicating immature neuronal microtubule structures. In summary, all three experimental treatment strategies were significantly more effective compared to the cyanoacrylate adhesive control group, significantly smaller scar tissues without ingrown vascular networks were formed, though, only mild antiviral properties of SiNP‐GF19 were observed.

Expression Dynamics Indicate Potential Roles of KIF17 for Nuclear Reshaping and Tail Formation during Spermiogenesis in Phascolosoma esculenta.

Kinesin family member17 (KIF17), a homologous dimer of the kinesin-2 protein family, has important microtubule-dependent and -independent roles in spermiogenesis. Little is known about KIF17 in the mollusk, Phascolosoma esculenta, a newly developed mariculture species in China. Here, we cloned the open reading frame of Pe-kif17 and its related gene, Pe-act, and performed bioinformatics analysis on both. Pe-KIF17 and Pe-ACT are structurally conserved, indicating that they may be functionally conserved. The expression pattern of kif17/act mRNA performed during spermiogenesis revealed their expression in diverse tissues, with the highest expression level in the coelomic fluid of P. esculenta. The expressions of Pe-kif17 and Pe-act mRNA were relatively high during the breeding season (July-September), suggesting that Pe-KIF17/ACT may be involved in spermatogenesis, particularly during spermiogenesis. Further analysis of Pe-kif17 mRNA via fluorescence in situ hybridization revealed the continuous expression of this mRNA during spermiogenesis, suggesting potential functions in this process. Immunofluorescence showed that Pe-KIF17 co-localized with α-tubulin and migrated from the perinuclear cytoplasm to one side of the spermatid, forming the sperm tail. Pe-KIF17 and Pe-ACT also colocalized. KIF17 may participate in spermiogenesis of P. esculenta, particularly in nuclear reshaping and tail formation by interacting with microtubule structures similar to the manchette. Moreover, Pe-KIF17 with Pe-ACT is also involved in nuclear reshaping and tail formation in the absence of microtubules. This study provides evidence for the role of KIF17 during spermiogenesis and provides theoretical data for studies of the reproductive biology of P. esculenta. These findings are important for spermatogenesis in mollusks.

Self-Rolled-Up Ultrathin Single-Crystalline Silicon Nanomembranes for On-Chip Tubular Polarization Photodetectors.

Freestanding single-crystalline nanomembranes and their assembly have broad application potential in photodetectors for integrated chips. However, the release and self-assembly process of single-crystalline semiconductor nanomembranes still remains a great challenge in on-chip processing and functional integration, and photodetectors based on nanomembrane always suffer from limited absorption of nanoscale thickness. Here, a non-destructive releasing and rolling process is employed to prepare tubular photodetectors based on freestanding single-crystalline Si nanomembranes. Spontaneous release and self-assembly are achieved by residual strain introduced by lattice mismatch at the epitaxial interface of Si and Ge, and the intrinsic stress and strain distributions in self-rolled-up Si nanomembranes are analyzed experimentally and computationally. The advantages of light trapping and wide-angle optical coupling are realized by tubular geometry. This Si microtube device achieves reliable Ohmic contact and exhibits a photoresponsivity of over 330mA W-1 , a response time of 370 µs, and a light incident detection angle range of over 120°. Furthermore, the microtubular structure shows a distinct polarization angle-dependent light absorption, with a dichroic ratio of 1.24 achieved at 940nm. The proposed Si-based microtubes provide new possibilities for the construction of multifunctional chips for integrated circuit ecosystems in the More than Moore era.

De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures invitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5-/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the invivo functions of Tektin 5.