Microtubule Remodeling Research Articles

Architecture-driven quantitative nanoscopy maps cytoskeleton remodeling

Cytoskeleton remodeling which generates force and orchestrates signaling and trafficking to govern cell migration remains poorly understood, partly due to a lack of an investigation tool with high system flexibility, spatiotemporal resolution, and computational sensitivity. Herein, we developed a multimodal superresolution imaging system-based architecture-driven quantitative (ADQ) framework in spatiotemporal-angular hyperspace to enable both identification of the optimal imaging mode with well-balanced fidelity and phototoxicity and accurate postcharacterization of microtubule remodeling. In the ADQ framework, a pixel/voxel-wise metric reflecting heterogeneous intertubule alignment was proposed with improved sensitivity over previous efforts and further incorporated with temporal features to map dynamic microtubule rearrangements. The ADQ framework was verified by assessing microtubule remodeling in drug-induced (de)polymerization, lysosome transport, and migration. Different remodeling patterns from two migration modes were successfully revealed by the ADQ framework, with a front-rear polarization for individual directed migration and a contact site-centered polarization for cell-cell interaction-induced migration in an immune response model. Meanwhile, these migration modes were found to have consistent orientation changes, which exhibited the potential of predicting migration trajectory.

Lactobacillus Restructures the Micro/Mycobiome to Combat Glycoprotein-130 Associated Microtubule Remodeling and Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

Emerging data demonstrate systemic and local inflammation regulate right ventricular (RV) adaption in preclinical and human pulmonary arterial hypertension (PAH). Pathological RV inflammation is targetable as antagonism of glycoprotein-130 (GP130) signaling counteracts pathological microtubule remodeling and improves RV function in rodents. Microtubules control several aspects of cardiomyocyte biology including cellular and nuclear size/structure, t-tubule homeostasis, and the proper localization of connexin-43. The intestinal microbiome regulates systemic inflammation, but the impact of the gut microbiome on the GP130-microtubule axis in RV failure is unknown. Here, we examined how the anti-inflammatory bacteria, Lactobacillus , modulated cellular and physiological RV phenotypes in preclinical and clinical PAH. Lactobacillus supplementation restructured the gut micro/mycobiome, suppressed systemic inflammation, combatted pathological GP130-mediated RV cardiomyocyte microtubule remodeling, and augmented RV function in rodent PAH. Moreover, Lactobacillus was associated with superior RV adaption in human PAH. These data further support the hypothesis that inflammation negatively impacts RV adaption in PAH, and identify the gut microbiome as a potentially targetable regulator of RV function in PAH.

Comparative transcriptome profiling reveals that light coordinates auxin to inhibit adventitious root formation in grapevine

Grapevine (Vitis sp.) is one of the most important economic fruit crops all over the world, and the formation of adventitious roots (ARs) is crucial for the vegetative reproduction of grapes. However, studies on the regulatory mechanisms of this process are currently lacking. In this study, we applied an efficient and convenient leave-petiole (LP) system for studying ARs, revealing a significant inhibition of root primordia formation under continuous-light treatment. The results showed that isolated ARs of grapevine were induced and originated from ray cells near the vascular cambium, with the process categorized into induction, initiation, and extension stages. LP samples under light and dark conditions were used for transcriptome sequencing and endogenous hormone measurements at three critical time points of AR formation. A total of 37 155 transcripts were obtained, and 7041 genes showed significantly different expression levels in the petiole. An integrated analysis, including Gene Ontology (GO) enrichment analysis, weighted gene co-expression network analysis (WGCNA), and hormonal content determination, showed that several genes (ARF4, LAX1, PIN1, SUS2, APX1, TPXL1, CHS3, etc.) associated with hormone signals, sugar synthesis and transport, reactive oxygen species (ROS) scavenging, cell wall biogenesis, flavonoid biosynthesis, microtubule remodeling, and some transcription factors (HY5, COP1, ERF2, MYB15, etc) played vital roles in light-induced AR formation. A hypothetical model was initially constructed, which illustrated the centrality of auxin in HY5-dependent AR formation and the complex crosstalk among various factors. The results of this study provided abundant genetic resources and a novel perspective for understanding the molecular mechanisms of AR formation in grapevine.

A divergent protein kinase A regulatory subunit essential for morphogenesis of the human pathogen Leishmania.

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote differentiation into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastidae. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of pkar3 or pkac3 null mutants lose their elongated shape and become rounded but remain flagellated. Truncation of an N-terminal formin homology (FH)-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARIβ and PKARIIβ binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 binds nucleoside analogs, but not cAMP, with a high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early-diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape.

Balancing Act: Tubulin Glutamylation and Microtubule Dynamics in Toxoplasma gondii.

The success of the intracellular parasite Toxoplasma gondii in invading host cells relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The T. gondii genome encodes three isoforms of both α- and β-tubulin, which undergo specific post-translational modifications (PTMs), altering the biochemical and biophysical proprieties of microtubules and modulating their interaction with associated proteins. Tubulin PTMs represent a powerful and evolutionarily conserved mechanism for generating tubulin diversity, forming a biochemical 'tubulin code' interpretable by microtubule-interacting factors. T. gondii exhibits various tubulin PTMs, including α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and β-tubulin polyglutamylation, and α- and β-tubulin methylation. Tubulin glutamylation emerges as a key player in microtubule remodeling in Toxoplasma, regulating stability, dynamics, interaction with motor proteins, and severing enzymes. The balance of tubulin glutamylation is maintained through the coordinated action of polyglutamylases and deglutamylating enzymes. This work reviews and discusses current knowledge on T. gondii tubulin glutamylation. Through in silico identification of protein orthologs, we update the recognition of putative proteins related to glutamylation, contributing to a deeper understanding of its role in T. gondii biology.

A cytoskeleton symphony: Actin and microtubules in microglia dynamics and aging

Microglia dynamically reorganize their cytoskeleton to perform essential functions such as phagocytosis of toxic protein aggregates, surveillance of the brain parenchyma, and regulation of synaptic plasticity during neuronal activity bursts. Recent studies have shed light on the critical role of the microtubule cytoskeleton in microglial reactivity and function, revealing key regulators like cyclin-dependent kinase 1 and centrosomal nucleation in the remodeling of microtubules in activated microglia. Concurrently, the role of the actin cytoskeleton is also pivotal, particularly in the context of small GTPases like RhoA, Rac1, and Cdc42 and actin-binding molecules such as profilin-1 and cofilin. This article delves into the intricate molecular landscape of actin and microtubules, exploring their synergistic roles in driving microglial cytoskeletal dynamics. We propose a more integrated view of actin and microtubule cooperation, which is fundamental to understanding the functional coherence of the microglial cytoskeleton and its pivotal role in propelling brain homeostasis. Furthermore, we discuss how alterations in microglial cytoskeleton dynamics during aging and in disease states could have far-reaching implications for brain function. By unraveling the complexities of microglia cytoskeletal dynamics, we can deepen our understanding of microglial functional states and their implications in health and disease, offering insights into potential therapeutic interventions for neurologic disorders.

Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2.

Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

Abstract 15932: Lactobacillus Suppresses Glycoprotein-130-Mediated Microtubule Remodeling to Support Right Ventricular Function in Pulmonary Arterial Hypertension

Introduction: Microtubules (MTs) are crucial for cardiomyocyte function as they regulate t-tubule structure, nuclear morphology, and gap junction protein localization. Glycoprotein-130 (GP130) signaling promotes pathological MT remodeling and right ventricular (RV) dysfunction in pulmonary arterial hypertension (PAH), but the trigger for the inflammation-MT connection is unknown. Microbiome dysbiosis activates systemic inflammation, and levels of the anti-inflammatory bacteria Lactobacillus are associated with RV function in rodents. However, the direct effects of Lactobacillus on GP130 signaling, MT remodeling, and RV function in PAH are unknown. Methods: Rats were randomly allocated into 3 groups: control, MCT-water, and MCT rats given Lactobacillus (MCT- Lactobacillus ). Echocardiography and pressure-volume loops defined RV function. Next-generation sequencing evaluated the composition of the bacterial components of the fecal microbiome. SomaScan proteomics quantified the serum proteome. RV and jejunal morphology were assessed with light and super resolution confocal microscopy. The relationship between Lactobacillus abundance and RV response to afterload was assessed in 65 PAH patients. Results: Lactobacillus augmented RV function (A) and reduced RV hypertrophy and fibrosis without altering PAH severity. Lactobacillus shifted the serum proteomic signature towards control (B) and reduced circulating GP130 ligands, normalized RV t-tubule architecture (C), MT density (D), and nucleus size (E), and prevented connexin-43 lateralization in RV cardiomyocytes (F). Lactobacillus restructured the microbiome, and concomitantly improved jejunal epithelial cell morphology with increased villus/microvillus length and glycocalyx thickness. PAH patients with fecal Lactobacillus had superior RV adaptation as afterload increased (G). Conclusion: Lactobacillus combats GP130-mediated MT remodeling to support RV function.

Polarized microtubule remodeling transforms the morphology of reactive microglia and drives cytokine release

Microglial reactivity is a pathological hallmark in many neurodegenerative diseases. During stimulation, microglia undergo complex morphological changes, including loss of their characteristic ramified morphology, which is routinely used to detect and quantify inflammation in the brain. However, the underlying molecular mechanisms and the relation between microglial morphology and their pathophysiological function are unknown. Here, proteomic profiling of lipopolysaccharide (LPS)-reactive microglia identifies microtubule remodeling pathways as an early factor that drives the morphological change and subsequently controls cytokine responses. We find that LPS-reactive microglia reorganize their microtubules to form a stable and centrosomally-anchored array to facilitate efficient cytokine trafficking and release. We identify cyclin-dependent kinase 1 (Cdk-1) as a critical upstream regulator of microtubule remodeling and morphological change in-vitro and in-situ. Cdk-1 inhibition also rescues tau and amyloid fibril-induced morphology changes. These results demonstrate a critical role for microtubule dynamics and reorganization in microglial reactivity and modulating cytokine-mediated inflammatory responses.

Organization of cortical microtubules in differentiated cells.

The microtubule cytoskeleton plays a critical role in a variety of cellular activities, and its structures and functions have been extensively studied. However, little is known about cell differentiation-related microtubule remodeling, its regulatory mechanisms, and its physiological functions. Recent studies have shown that microtubule-binding proteins as well as cell junctions, such as desmosomes and adherens junctions, are involved in the remodeling of microtubules in response to cell differentiation. In addition, the microtubule-organizing activity and structural integrity of centrosomes undergo dramatic changes during cell differentiation to promote microtubule remodeling. Here we summarize recent advances revealing the dynamic changes in microtubule organization and functions during cell differentiation. We also highlight the molecular mechanisms underlying microtubule modeling in differentiated cells, focusing on the key roles played by microtubule-binding proteins, cell junctions, and centrosomes. This article is protected by copyright. All rights reserved.

CAMSAP2 localizes to the Golgi in islet β-cells and facilitates Golgi-ER trafficking

Glucose stimulation induces the remodeling of microtubules, which potentiates insulin secretion in pancreatic β-cells. CAMSAP2 binds to microtubule minus ends to stabilize microtubules in several cultured clonal cells. Here, we report that the knockdown of CAMSAP2 in primary β-cells reduces total insulin content and attenuates GSIS without affecting the releasability of insulin vesicles. Surprisingly, CAMSAP2 knockdown does not change microtubule stability. Unlike in cultured insulinoma cells, CAMSAP2 in primary β-cells predominantly localizes to the Golgi apparatus instead of microtubule minus ends. This novel localization is specific to primary β- but not α-cells and is independent of microtubule binding. Consistent with its specific localization at the Golgi, CAMSAP2 promotes efficientGolgi-ER trafficking in primary β-cells. Moreover, primary β-cells and insulinoma cells likely express different CAMSAP2 isoforms. We propose that a novel CAMSAP2 isoform in primary β-cells has a non-canonical function, which promotes Golgi-ER trafficking to support efficient production of insulin and secretion.

Identification of VASH1 as a Potential Prognostic Biomarker of Lower-Grade Glioma by Quantitative Proteomics and Experimental Verification.

VASH1 is a novel angiogenic regulatory factor, that participates in the process of carcinogenesis and the development of diverse tumors. Our study aimed to investigate the expression and prognostic value of the VASH1 in Lower-Grade Glioma (LGG), to explore its functional network in LGG and its effects on biological behaviors. LGG transcriptome data, somatic mutation profiles and clinical features analyzed in the present study were obtained from the TCGA, GTEx, CCLE, CGGA, UALCAN, and GEPIA2 databases, as well as clinical data and tissue sections of 83 LGG patients in our hospital. The expression characteristics of VASH1 in LGG were investigated by univariate, multivariate, immunohistochemistry, qRT-PCR, and western-blot. Subsequently, we analyzed the prognostic significance of VASH1 in LGG patients by survival analysis, subject operation characteristic curve, correlation analysis, external validation, independent prognostic significance analysis, and clinical stratification, and confirmed its biological effect on glioma cell lines in vitro. Finally, we performed GO, KEGG, and GSEA to clarify biological functions and related pathways. CIBERSORT and ESTIMATE algorithms were used to calculate the proportion of immune cells and immune microenvironment fraction in LGG. We found that VASH1 is highly expressed in LGG tissues and is associated with poor prognosis, WHO grade, IDH1 wild-type, and progressive disease (P < 0.05). Multivariate and the Nomogram model showed that high VASH1 expression was an independent risk factor for glioma prognosis and had better prognostic prediction efficacy in different LGG Patient cohorts (HR = 4.753 and P=0.002). In vitro experiments showed that knockdown of VASH1 expression in glioma cell lines caused increased glioma cell proliferation, invasion, and migration capacity. The mechanism may be related to VASH1 promoting microtubule formation and remodeling of immune microenvironment. Our study firstly found that high VASH1 expression was associated with poor prognosis. In addition, We identified the possible mechanism by which VASH1 functioned in LGG. VASH1 inhibits the invasion and migration of tumor cells by affecting microtubule formation and immune infiltration in the tumor microenvironment. May be an important endogenous anti-tumor factor for LGG and provide a potential biomarker for individualized treatment of LGG.

Morphogenesis Dynamics in Leishmania Differentiation.

Leishmania, the causative agent of leishmaniasis, is an obligatory intracellular parasite that cycles between phagolysosome of mammalian macrophages, where it resides as round intracellular amastigotes, and the midgut of female sandflies, where it resides as extracellular elongated promastigotes. This protozoan parasite cytoskeleton is composed of stable and abundant subpellicular microtubules (SPMT). This study aims to determine the kinetics of developmental morphogenesis and assess whether microtubules remodelling is involved in this process. Using image-streaming technology, we observed that rounding of promastigotes during differentiation into amastigotes was initiated promptly after exposure to the differentiation signal. Stabilizing microtubules with taxol sped rounding, but later killed differentiating parasites if taxol was not removed. Microtubule destabilizers such as vinblastine had no effect on the rate of rounding, nor on the viability of differentiating parasites. In the reverse process, elongation is initiated after a delay of 7.5 and completed 72 h after exposure to the amastigote to the promastigote differentiation signal. During the delay, parasites became highly sensitive to treatment with microtubule destabilizers. The addition of vinblastine during the first 7.5 h halted differentiation and killed parasites. Between 8 and 24 h, parasites gradually became resistant to vinblastine and, in parallel, started to elongate. In contrast, taxol had no effect on parasite elongation, nor on the viability of these cells. In a parallel study, we showed that the Leishmania-specific protein kinase A (PKA) holoenzyme containing the LdPKAR3-C3 complex is essential for promastigote elongation. Mutant promastigotes lacking either of these proteins are round, but maintain their flagella. Here, we observed that during differentiation into amastigotes, these mutants round at the same rate as the wild type, but never exceed the WT density of round amastigotes. In the reverse process, these mutants undergo the same initial delay and then elongate at the same rate as the WT. They stop elongating when they reach 20% of elongated cells in mature promastigotes. Our analysis indicates that while promastigote rounding into amastigotes did not require microtubule remodelling, morphogenesis of round amastigotes into elongated promastigotes required microtubule rearrangement before elongation was initiated. This is the first study that investigates the dynamics of microtubules during parasite development.

Hypergravity Attenuates Reactivity in Primary Murine Astrocytes.

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approaches.

The Chemerin/CMKLR1 Axis Is Involved in the Recruitment of Microglia to Aβ Deposition through p38 MAPK Pathway.

The accumulation of microglia around senile plaques is one of the pathological features of Alzheimer’s disease (AD). Chemerin is an adipokine with immune-modulating properties. Our previous study showed that chemokine-like receptor 1 (CMKLR1), the receptor for chemerin, is also a functional receptor of Aβ. However, it remains unclear whether and how the chemerin/CMKLR1 axis affects the migration of microglia. The impact of CMKLR1 on microglial activation and recruitment toward Aβ deposits was examined in APP/PS1 mice mated with CMKLR1 knockout (CMKLR1−/−) mice. CMKLR1 deficiency reduced the number of microglia around Aβ deposits in aged APP/PS1-CMKLR1−/− mice compared with APP/PS1 mice. Chemerin expression was significantly decreased in the hippocampus and cortex of aged APP/PS1 mice compared with WT mice. In vitro assays demonstrated that activation of the chemerin/CMKLR1 axis promoted the migration of primary cultures of microglia and murine microglial N9 cells. Mechanistic studies found that chemerin/CMKLR1 induced polarization and protrusion formation of microglia by promoting the remodeling of actin filaments and microtubules, and Golgi apparatus reorientation. The inhibition of p38 MAPK attenuated the promotion of the chemerin/CMKLR1 axis on microglial migration and polarization. In addition, chemerin inhibited Aβ-induced microglial clustering. The inhibition of p38 MAPK alleviated the suppressive effect of chemerin on Aβ-induced microglial aggregation. Our data indicate that the chemerin/CMKLR1 axis is involved in the migration and recruitment of microglia to senile plaques via the p38 MAPK pathway. Modulation of the chemerin/CMKLR1 axis is a potential new strategy for AD therapy.

Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha.

Gametogenesis is an essential event for sexual reproduction in various organisms. Bryophytes employ motile sperm (spermatozoids) as male gametes, which locomote to the egg cells to accomplish fertilization. The spermatozoids of bryophytes harbor distinctive morphological characteristics, including a cell body with a helical shape and two flagella. During spermiogenesis, the shape and cellular contents of the spermatids are dynamically reorganized. However, the reorganization patterns of each organelle remain obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to changes in cellular and nuclear shapes and flagellar development. We then examined the remodeling of microtubules and the reorganization of endomembrane organelles. The results indicated that the state of glutamylation of tubulin changes during formation of the flagella and spline. We also found that the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner, which involves the functions of endosomal sorting complexes required for transport (ESCRT) machineries in endocytic and vacuolar transport. These findings are expected to provide useful indices to classify developmental and subcellular processes of spermiogenesis in bryophytes.

Common genetic variation in KATNAL1 non-coding regions is involved in the susceptibility to severe phenotypes of male infertility.

BackgroundPrevious studies in animal models evidenced that genetic mutations of KATNAL1, resulting in dysfunction of its encoded protein, lead to male infertility through disruption of microtubule remodelling and premature germ cell exfoliation. Subsequent studies in humans also suggested a possible role of KATNAL1 single‐nucleotide polymorphisms in the development of male infertility as a consequence of severe spermatogenic failure.ObjectivesThe main objective of the present study is to evaluate the effect of the common genetic variation of KATNAL1 in a large and phenotypically well‐characterised cohort of infertile men because of severe spermatogenic failure.Materials and methodsA total of 715 infertile men because of severe spermatogenic failure, including 210 severe oligospermia and 505 non‐obstructive azoospermia patients, as well as 1058 unaffected controls were genotyped for three KATNAL1 single‐nucleotide polymorphism taggers (rs2077011, rs7338931 and rs2149971). Case–control association analyses by logistic regression assuming different models and in silico functional characterisation of risk variants were conducted.ResultsGenetic associations were observed between the three analysed taggers and different severe spermatogenic failure groups. However, in all cases, the haplotype model (rs2077011*C | rs7338931*T | rs2149971*A) better explained the observed associations than the three risk alleles independently. This haplotype was associated with non‐obstructive azoospermia (adjusted p = 4.96E‐02, odds ratio = 2.97), Sertoli‐cell only syndrome (adjusted p = 2.83E‐02, odds ratio = 5.16) and testicular sperm extraction unsuccessful outcomes (adjusted p = 8.99E‐04, odds ratio = 6.13). The in silico analyses indicated that the effect on severe spermatogenic failure predisposition could be because of an alteration of the KATNAL1 splicing pattern.ConclusionsSpecific allelic combinations of KATNAL1 genetic polymorphisms may confer a risk of developing severe male infertility phenotypes by favouring the overrepresentation of a short non‐functional transcript isoform in the testis.

P-538 KATNAL1 polymorphisms confer susceptibility to severe phenotypes of male infertility in a large European cohort

Abstract Study question What is the contribution of the common variation of KATNAL1 to the development of severe spermatogenic failure (SPGF) in a phenotypically well-characterized cohort? Summary answer An allelic combination of KATNAL1 single-nucleotide polymorphisms (SNPs) increases the risk to develop SPGF, likely by altering the expression and splicing pattern of the gene. What is known already Spermatogenesis is a process that requires an exhaustive control of gene expression, and subtle alterations of its molecular regulatory network can lead to male infertility. The aetiology of most SPGF cases remains unknown, and increasing evidence clearly suggests that the idiopathic form of SPGF represents a complex trait, in which genetic susceptibility is conferred by the accumulation of risk alleles in genetically predisposed men. In this regard, previous studies reported that rare genetic mutations and polymorphisms in the KATNAL1 locus lead to male infertility through the disruption of microtubule remodelling and premature germ cell exfoliation. Study design, size, duration We designed a case-control genetic association study including three SNPs (rs2077011, rs7338931, and rs2149971) in the 3' and 5' regulatory regions of KATNAL1, which tagged the common genetic variability in the region. The allele frequencies in the study population, composed of 715 infertile men diagnosed with idiopathic SPGF, were compared to those observed in a control group comprising 1058 fertile men from Spain and Portugal. Participants/materials, setting, methods The SPGF group comprised 210 men with severe oligospermia (SO) and 505 with non-obstructive azoospermia (NOA). The latter were phenotypically characterised according to the histological examination of testis biopsies and its outcome (Sertoli cell-only syndrome, SCO; maturation arrest, MA; hypospermatogenesis, HS; and testicular sperm extraction, TESE). After genotyping, case-control association analyses by logistic regression were conducted. In silico functional characterization of risk variants was also carried out using public multiomic databases and bioinformatic tools. Main results and the role of chance Significant genetic associations with different SPGF patterns and/or TESE outcome were observed even after correction for multiple testing when independent SNP models were tested. However, in all cases, the haplotype model including the three risk alleles (rs2077011*C | rs7338931*T | rs2149971*A) was the most informative. This haplotype was specifically over-represented in the SPGF group (P = 3.45E-02, OR = 2.33), which includes all infertile men, and in the NOA group (P = 8.22E-03, OR = 2.97). In addition, subtype-specific associations were observed with the most severe subgroups, namely MA (P = 2.44E-02, OR = 5.00), SCO (P = 4.03E-03, OR = 5.16), and unsuccessful TESE outcome (P = 2.22E-04, OR = 6.13), which indicates the relevant role of KATNAL1 in spermatogenesis development. We prioritized the most likely causal variant/s based on in silico analyses addressing the possible functional implication of the tagged variants. We observed that an alteration of the KATNAL1 splicing pattern, by favouring the overrepresentation of a short non-functional transcript isoform in the testis, might be the cause behind the observed genetic association. The analysis of the testis transcriptome at the single cell level showed that KATNAL1 transcripts were mostly presented in spermatocytes and early spermatids at puberty, which correlates with its effect of premature exfoliation and loss of the germ cells. Limitations, reasons for caution Although a previous low-powered study reported suggestive associations between KATNAL1 and SPGF, additional genetic association studies in independent populations should be conducted to confirm our findings. Moreover, the statistical power for the overall analysis was appropriate, but the subphenotype analyses were performed with reduced power due to smaller study groups. Wider implications of the findings Our results suggest a relevant role of the common genetic variation of KATNAL1 in the susceptibility to develop the most severe histological phenotypes of NOA (i.e., SCO and MA). Studies like the one presented here may definitively help to develop future non-invasive molecular markers of TESE success. Trial registration number Not Applicable

Re-evaluating the actin-dependence of spectraplakin functions during axon growth and maintenance.

Axons are the long and slender processes of neurons constituting the biological cables that wire the nervous system. The growth and maintenance of axons require loose microtubule bundles that extend through their entire length. Understanding microtubule regulation is therefore an essential aspect of axon biology. Key regulators of neuronal microtubules are the spectraplakins, a well‐conserved family of cytoskeletal cross‐linkers that underlie neuropathies in mouse and humans. Spectraplakin deficiency in mouse or Drosophila causes severe decay of microtubule bundles and reduced axon growth. The underlying mechanisms are best understood for Drosophila’s spectraplakin Short stop (Shot) and believed to involve cytoskeletal cross‐linkage: Shot's binding to microtubules and Eb1 via its C‐terminus has been thoroughly investigated, whereas its F‐actin interaction via N‐terminal calponin homology (CH) domains is little understood. Here, we have gained new understanding by showing that the F‐actin interaction must be finely balanced: altering the properties of F‐actin networks or deleting/exchanging Shot's CH domains induces changes in Shot function—with a Lifeact‐containing Shot variant causing remarkable remodeling of neuronal microtubules. In addition to actin‐microtubule (MT) cross‐linkage, we find strong indications that Shot executes redundant MT bundle‐promoting roles that are F‐actin‐independent. We argue that these likely involve the neuronal Shot‐PH isoform, which is characterized by a large, unexplored central plakin repeat region (PRR) similarly existing also in mammalian spectraplakins.

Telomere shortening impairs alveolar regeneration.

ObjectivesShort telomeres in alveolar type 2 (AT2) cells have been associated with many lung diseases. The study aimed to investigate the regeneration capacity of AT2 cells with short telomeres by knocking out Tert in mice (G4 Tert −/− ) from the whole to the cellular level.Materials and MethodsThe lung injury model of mice was established by left pneumonectomy (PNX). The proliferation and differentiation of AT2 cells were observed by immunofluorescence staining in vivo and in vitro. The difference of the gene expression between control and G4 Tert −/− group during the regeneration of AT2 cells was compared by RNA sequencing. The expression of tubulin polymerization promoting protein 3 (TPPP3) was reduced by adeno‐associated virus delivery.ResultsThe alveolar regeneration in G4 Tert −/− mice was impaired after PNX‐induced lung injury. The regulation of cytoskeleton remodelling was defective in G4 Tert −/− AT2 cells. The expression of TPPP3 was gradually increased during AT2 cell differentiation. The expression level of TPPP3 was reduced in G4 Tert −/− AT2 cells. Reducing TPPP3 expression in AT2 cells limits the microtubule remodelling and differentiation of AT2 cells.ConclusionShort telomeres in AT2 cells result in the reduced expression level of TPPP3, leading to impaired regeneration capacity of AT2 cells.