Peripheral Microtubules Research Articles

B cell mechanosensing regulates ER remodeling at the immune synapse.

Engagement of the B-cell receptor with immobilized antigens triggers the formation of an immune synapse (IS), a complex cellular platform where B-cells recruit signaling molecules and reposition lysosomes to promote antigen uptake and processing. Calcium efflux from the endoplasmic reticulum (ER) released upon BCR stimulation is necessary to promote B-cell survival and differentiation. Whether the spatial organization of the ER within the B-cell synapse can tune IS function and B-cell activation remains unaddressed. Here, we characterized ER structure and interaction with the microtubule network during BCR activation and evaluated how mechanical cues arising from antigen presenting surfaces affect this process. B-cells were cultured on surfaces of varying stiffness coated with BCR ligands, fixed, and stained for the ER and microtubule network. Imaging analysis was used to assess the distribution of the ER and microtubules at the IS. Upon BCR activation, the ER is redistributed towards the IS independently of peripheral microtubules and accumulates around the microtubule-organization center. Furthermore, this remodeling is also dependent on substrate stiffness, where greater stiffness triggers enhanced redistribution of the ER. Our results highlight how spatial reorganization of the ER is coupled to the context of antigen recognition and could tune B-cell responses. Additionally, we provide novel evidence that the structural maturation of the ER in plasma cells is initiated during early activation of B-cells.

CCDC189 depletion leads to oligo-astheno-teratozoospermia and male infertility in mice†.

In male reproductive system, proteins containing the coiled-coil domain (CCDC) are predominantly expressed in specific regions including the testis, epididymis, seminal vesicle, and prostate. They play a vital role in centriole formation, sperm motility and flagellar development in male gametes. Despite being highly expressed in the testis, the exact physiological function of the coiled-coil domain-containing 189 (Ccdc189) gene remain largely unclear. Our research provides a comprehensive and detailed investigation into the localization of CCDC189 protein within the testis seminiferous tubules. CCDC189 specifically expressed in spermatocytes, round spermatids, and elongating spermatids in mouse testis. The deletion of Ccdc189 in mouse leads to male infertility, characterized by significantly reduced sperm counts and motility. Abnormally shaped spermatozoa with irregular tails, exhibiting shortened and twisted morphology, were observed in the seminiferous tubules. Electron microscopy revealed disordered and missing peripheral microtubule doublets (MTD) and outer dense fibers (ODF) in the sperm flagella, accompanied by a consistent absence of central pairs (CP). The knockout of Ccdc189 resulted in oligo-astheno-teratozoospermia, which is characterized by low sperm count and reduced sperm motility and abnormal morphology. Furthermore, we identified poly(A)-binding protein cytoplasmic 1 (PABPC1) and PABPC2 as interacting proteins with CCDC189. These proteins belong to the PABP family and are involved in regulating mRNA translational activity in spermatogenic cells by specifically binding to poly(A) tails at the 3' ends of mRNAs.

P-154 Structural changes of the human sperm tail during the early post-fertilization

Abstract Study question What happens to the sperm tails that are no longer needed after the goal of fertilization has been achieved? Summary answer The sperm tail lost its fibrous sheath and changed to a coiled morphology with a whisker root-like structure at the tip during the early post-fertilization. What is known already The sperm tail has been thought to play only a role in allowing the head and neck to reach the oocyte. The flagellum is found in the mammalian sperm tail and consists of a middle, main, and terminal part. It also contains an axon composed of two central microtubules surrounded by nine peripheral microtubule doublets (9d + 2s microtubule pattern) surrounded by an accessory structure called the fibrous sheath (FS), but few studies have been conducted on the post-fertilization fate of the mammalian sperm tail. Study design, size, duration To investigate sperm tail structure from the pronuclear stage to the second cleavage, we used abnormally fertilized oocytes derived from either conventional in vitro fertilization (cIVF, n = 41) or intracytoplasmic sperm injection (ICSI, n = 11). In this study, abnormally fertilized oocytes were collected between March 2022 and November 2023 and subjected to immunofluorescence analysis and field emission scanning electron microscopy (FE-SEM) analysis. Participants/materials, setting, methods Under ethical approval, abnormally fertilized oocytes were donated by infertile couples undergoing cIVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. Informed consent was obtained from all participants. Immunofluorescence assay was performed to analyze sperm tail morphology using antibodies against H3K27me3, α-tubulin, and AKAP4. FE-SEM analysis was performed to detect the 9d + 2s structure characteristic of the sperm tail axon. Main results and the role of chance Tri-pronuclear zygotes derived from conventional in vitro fertilization (cIVF-derived 3PN) had two male pronuclei, whereas those derived from intracytoplasmic sperm injection (ICSI-derived 3PN) had one. Each zygote had the same number of coiled microtubule structures as the male pronucleus, and the 9d + 2s structure characteristic of the axon was identified, suggesting a sperm tail. No difference in sperm tail shape was observed between ICSI-derived 3PN and cIVF-derived 3PN. AKAP4, which is specifically localized in the FS associated with the principal piece of the flagellum, was observed in sperm tails attached to the zona pellucida but not in those in the oocyte, suggesting that the FS of the flagellum is lost before the pronuclear stage, leaving the microtubules of the axonemes exposed. The sperm tail was attached to one of the spindle poles during the first cleavage of abnormally fertilized oocytes, but no differences in the structure of the spindle pole with or without the sperm tail were observed. The tip of the sperm tail was branched like a “whisker root”, suggesting that the sperm tail was degraded from the end, but the degree of tip branching did not differ from the pronuclear stage to the second cleavage. Limitations, reasons for caution Normal fertilized oocytes were not used in this study because they are used for clinical treatment. The cell biological significance of sperm tail branching is currently unknown and requires further experimentation. Wider implications of the findings These findings shed light on the processes that occur during human fertilization, suggest new roles for the sperm tail, and may have implications for assisted fertilization approaches in the future. Trial registration number not applicable

A splice donor variant of GAS8 induces structural disorganization of the axoneme in sperm flagella and leads to nonsyndromic male infertility.

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.

LRRC23 deficiency causes male infertility with idiopathic asthenozoospermia by disrupting the assembly of radial spokes.

Asthenozoospermia (AZS) is the primary cause of infertility in males. The radial spoke (RS) is an axonemal structure, connecting the peripheral doublet microtubules with the central pair of microtubules. This T-shaped multiprotein complex functions as a mechanochemical sensor to promote sperm motility. LRRC23 is a novel subunit of the RS complex that is necessary for flagellar assembly and movement in mice. However, the importance of LRRC23 in modulating RS formation in humans remains unclear. Here, we identified a homozygous nonsense mutation in LRRC23 (c.376C>T:p. Arg126X) in an infertile AZS patient whose parents were consanguineous. We verified the adversity of this novel mutation because of its ability to disrupt LRRC23 synthesis and impair RSs integrity. Furthermore, we demonstrated an interaction between LRRC23 and RSPH3 in vitro, indicating that LCCR23 is associated with RS in humans. Meanwhile, the LRRC23-mutant patient had a good prognosis following intracytoplasmic sperm injection. This study provides strong preliminary evidence that LRRC23 defects are potential causative factors of AZS in humans, which expands our knowledge for improved genetic counseling and better reproductive recommendations for patients with AZS.

CCDC189 affects sperm flagellum formation by interacting with CABCOCO1.

Multiple morphological abnormalities of the sperm flagella (MMAF) are one of the major causes of male infertility and are characterized by multiple defects. In this study, we found that the coiled-coil domain-containing 189 (Ccdc189) gene was predominantly expressed in mouse testes and that inactivation of the Ccdc189 gene caused male infertility. Histological studies revealed that most sperm from Ccdc189-deficient mice carried coiled, curved or short flagella, which are typical MMAF phenotypes. Immunoelectron microscopy showed that the CCDC189 protein was located at the radial spoke of the first peripheral microtubule doublet in the sperm axoneme. A CCDC189-interacting protein, CABCOCO1 (ciliary-associated calcium-binding coiled-coil protein 1), was discovered via co-immunoprecipitation and mass spectrometry, and inactivation of Cabcoco1 caused malformation of sperm flagella, which was consistent with findings obtained with Ccdc189-deficient mice. Further studies revealed that inactivation of CCDC189 caused downregulation of CABCOCO1 protein expression and that both CCDC189 and CABCOCO1 interacted with the radial-spoke-specific protein RSPH1 and intraflagellar transport proteins. This study demonstrated that Ccdc189 is a radial-spoke-associated protein and is involved in sperm flagellum formation through its interactions with CABCOCO1 and intraflagellar transport proteins.

P-720 Biallelic variants in IQCN cause sperm flagella assembly defects and male infertility

Abstract Study question What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Summary answer Deficiency in IQCN is responsible for sperm flagella assembly defects and abnormal computer-assisted sperm analysis (CASA) parameters, which result in male infertility. What is known already Manchette is a transient structure that is involved in the shaping of the spermatid nucleus and protein transport of flagella. Most recently, our group reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN led to total fertilization failure (TFF) and defective acrosome structure phenotypes. However, the related function of IQCN in sperm flagella assembly is still unknown. Study design, size, duration Fifty infertile males were recruited from the Reproductive and Genetic Hospital of China International Trust Investment Corporation (CITIC)-Xiangya from January 2014 to October 2022. Participants/materials, setting, methods Genomic DNA was extracted from the peripheral blood samples of the affected individuals for whole-exome sequencing. The ultrastructure of the flagella was detected by transmission electron microscopy (TEM). CASA was used to test the parameters of sperm motility. An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPER-Cas9 technology. Immunoprecipitation (IP) followed by liquid chromatography‒mass spectrometry was used to select the differentially expressed IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Main results and the role of chance Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our infertile cohort. The sperm from the affected individuals showed irregular “9 + 2” structure of sperm flagella, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. The CASA parameters in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. The partial of peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or the chaotic arrangement of DMTs were observed in the principal piece and end piece of sperm flagella. The hyperactivation ability of sperm from Iqcn-/- male mice was decreased than sperm from Iqcn+/+ male mice. The CDC42 and IFT protein families are hub proteins that interact with IQCN and regulate flagella assembly during spermiogenesis. Deficiency in IQCN causes the loss of binding with CDC42 and IFT74, which leads to the absence or abnormal localization of CDC42 and IFT74 in mouse and human spermatozoa. Limitations, reasons for caution More cases are needed to demonstrate the relationship between the IQCN variations and the phenotypes. Wider implications of the findings Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, thus providing a genetic marker for sperm motility deficiency and male infertility. Trial registration number N.A.

Fine ultrastructural features of germ cells and spermatozoa during spermatogenesis in the European grayling, Thymallus thymallus (Teleostei, Salmoniformes, Salmonidae).

This study aimed to examine the ultrastructure of spermatogenic stages and mature spermatozoa in the European grayling, Thymallus thymallus. The testes were examined microscopically with a transmission electron microscope to find out details of the structure and morphology of the grayling germ cells, spermatozoa and some somatic cells. The grayling testis has a tubular shape, with cysts or clusters of germ cells within seminiferous lobules. The spermatogenic cells, including spermatogonia, spermatocytes, and spermatids, can be found along seminiferous tubules. There are electron-dense bodies in germ cells from the primary spermatogonia to secondary spermatocyte stages. These undergo mitosis to reach the secondary spermatogonia stage, when they form primary and secondary spermatocytes. Spermatids undergo three different stages of differentiation during spermiogenesis, characterized by the level of chromatin condensation, elimination of cytoplasm, and the occurrence of the flagellum. The midpiece of spermatozoa is short and contains spherical or ovoid mitochondria. The sperm flagellum has an axoneme with nine doublets of peripheral microtubules and two central microtubules. The result of this study is valuable to be used as a standard reference for germ cell development, which is of great importance to get a clear insight into the process of grayling breeding practice.

Biallelic variants in IQCN cause sperm flagellar assembly defects and male infertility.

What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPR-Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography-mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. VSL, VCL, and VAP in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn-/- male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. More cases are needed to demonstrate the relation between IQCN variants and phenotypes. Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. N/A.

Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

Midzone microtubule dynamics in anaphase cells.

During anaphase, antiparallel overlapping midzone microtubules elongate, contributing to chromosome segregation and specify of the location where the contractile ring will form. Midzone microtubules are dynamic in early anaphase, but not in late anaphase, when they appear to be stabilized. The kinetics and mechanisms of this stabilization are not completely understood. Here, we quantify of midzone microtubule dynamics using a photoactivatable-GFP tubulin expressed in LLCPk1 cells. We find that immediately after anaphase onset, a single highly dynamic population of microtubules is present, but as anaphase progresses, both a dynamic and more stable population of microtubules coexist, with a gradual increase in the dissipation half-time for the slower population. By the middle of cytokinesis, only static microtubules are detected. We examined the microtubule dynamics during furrowing examining the effects of actin and midzone-associated proteins. Blocking furrow ingression using either C3 or latrunculin treatment resulted in two spatially distinct microtubule populations in telophase cells: peripheral microtubules that were highly dynamic and central microtubules with fast and slow populations that never transition to a static array. Depletion of midzone microtubule-associate proteins, PRC1 or Kif4a, did not block furrow ingression in most cells and prevented formation of a static array in telophase cells. Although microtubules were partially stabilized similar to mid-anaphase cells. Quantification of the length of PRC1 decorated microtubule overlaps showed that static arrays are characterized by short highly compacted zones. These results demonstrate that dynamic turnover and sliding of midzone microtubules is gradually reduced as anaphase progresses and microtubules are further stabilized during cytokinesis. These data reveal the importance of the PRC1/KIF4A module and midzone microtubule compaction in generating a static midzone.

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules

The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47−/− ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.

Peripheral astral microtubules ensure asymmetric furrow positioning in neural stem cells.

Neuroblast division is characterized by asymmetric positioning of the cleavage furrow, resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly are governed by centralspindlin that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In neuroblasts, these two centralspindlin populations are spatially and temporally separated. A leading pool is located at the basal cleavage site and a second pool accumulates at the midzone before traveling to the cleavage site. The cortical centralspindlin population requires peripheral astral microtubules and the chromosome passenger complex for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin signaling at the midzone, leading to equatorial furrow repositioning and decreased size asymmetry. These data show that basal furrow positioning in neuroblasts results from a competition between different centralspindlin pools in which the cortical pool is dominant.

Ultrastructural features of aporocotylid blood flukes: The tegument and sensory receptors of Sanguinicola inermis Plehn, 1905 from the pike Esox lucius, with a comparative analysis of their traits within the Neodermata

This work represents the first ultrastructural study of the tegument of a freshwater teleost-infecting aporocotylid blood fluke and the first detailed observations of the surface sensory receptors of any aporocotylid. The work was carried out on Sanguinicola inermis from the branchial arteries of the pike, Esox lucius. Light and scanning electron microscopy observations failed to show the presence of protruding surface spines in S. inermis. Transmission electron microscopy studies revealed, deep beneath the distal cytoplasm of the tegument, sporadic, membrane-bound sarcoplasmic processes, each containing a single spine with the typical crystalline structure of digenean spines. On rare occasions, the spine may protrude above the body surface, but in such cases the distal tegumental cytoplasm is extremely flattened. This finding supports a unique, deep origin of aporocotylid spines when compared to those of other digenean families. This character may well prove to be an autapomorphy of the Aporocotylidae, and the position of the base of aporocotylid spines within sarcoplasmic processes supports their origin as a muscle derivative. Unlike other digenean groups, the distal region of the cytoplasmic processes, connecting sunken tegumental perikarya with the syncytial tegumental cytoplasm, is strengthened by peripheral microtubules. Three types of ciliated and three types of non-ciliated sensory receptors were distinguished in the tegument of S. inermis. One of the distinctive features of the ciliated receptors is the unusual position of the basal region of the cilium within a tegumental outgrowth. Such a position has previously been described for certain receptors types in larval and adult schistosomatids and larval diplostomids, suggesting that this feature, in adult worms, might prove to be an apomorphic trait for some or all groups of blood flukes.

Betaine Ameliorates Schizophrenic Traits by Functionally Compensating KIF3-Based CRMP2 Transport

Neurons in schizophrenia (SCZ) brains tend to undergo gross morphological changes, but its molecular mechanism is still largely elusive. Using Kif3b+/- mice as a SCZ model, here we show that high betaine diet can significantly remedy their schizophrenic traits in neuronal morphogenesis and social behavior. We identified significant reduction and restoration by betaine of lamellipodial dynamics in developing Kif3b+/- neurons as a cause of neurite hyperbranching, according to a local deficiency of collapsin response mediator protein 2 (CRMP2) transported by the KIF3 motor. Betaine administration significantly decreased the CRMP2 carbonylation, which enhanced its F-actin bundling activity necessary for proper lamellipodial dynamics and peripheral microtubule exclusion, and thus may functionally compensate the KIF3 deficiency. Because KIF3 expression levels tended to be downregulated in human prefrontal cortex of postmortem SCZ patient brains, this mechanism may also take part in human SCZ pathogenesis, which we propose will be remedied by betaine administration.

Glucose Regulates Microtubule Disassembly and the Dose of Insulin Secretion via Tau Phosphorylation.

The microtubule cytoskeleton of pancreatic islet β-cells regulates glucose-stimulated insulin secretion (GSIS). We have reported that the microtubule-mediated movement of insulin vesicles away from the plasma membrane limits insulin secretion. High glucose-induced remodeling of microtubule network facilitates robust GSIS. This remodeling involves disassembly of old microtubules and nucleation of new microtubules. Here, we examine the mechanisms whereby glucose stimulation decreases microtubule lifetimes in β-cells. Using real-time imaging of photoconverted microtubules, we demonstrate that high levels of glucose induce rapid microtubule disassembly preferentially in the periphery of individual β-cells, and this process is mediated by the phosphorylation of microtubule-associated protein tau. Specifically, high glucose induces tau hyper-phosphorylation via glucose-responsive kinases GSK3, PKA, PKC, and CDK5. This causes dissociation of tau from and subsequent destabilization of microtubules. Consequently, tau knockdown in mouse islet β-cells facilitates microtubule turnover, causing increased basal insulin secretion, depleting insulin vesicles from the cytoplasm, and impairing GSIS. More importantly, tau knockdown uncouples microtubule destabilization from glucose stimulation. These findings suggest that tau suppresses peripheral microtubules turning over to restrict insulin oversecretion in basal conditions and preserve the insulin pool that can be released following stimulation; high glucose promotes tau phosphorylation to enhance microtubule disassembly to acutely enhance GSIS.

CCDC61/VFL3 Is a Paralog of SAS6 and Promotes Ciliary Functions

SummaryCentrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.

Sperm characters of the aspidogastrean Rohdella amazonica (Aspidogastridae, Rohdellinae), a parasite of the banded puffer fish Colomesus psittacus.

The ultrastructural characteristics of the mature spermatozoon of the aspidogastrean Rohdella amazonica (Aspidogastridae, Rohdellinae) were studied by means of transmission electron microscopy. The sperm cell shows two axonemes of the 9 + '1' trepaxonematan pattern of Platyhelminthes, parallel cortical microtubules, a well-developed lateral expansion, external ornamentation of the plasma membrane, one mitochondrion, an electron-dense ring, a nucleus and granules of glycogen. The present results were compared with those observed in the aspidogastreans studied to date and in other Platyhelminthes. The lateral expansion and the electron-dense ring are typical characters for aspidogastreans. Although a lateral expansion has been described in other Platyhelminthes such as monogeneans and digeneans, the Aspidogastrea shows a much larger lateral expansion with both peripheral and internal microtubules. The dense ring is observed as a cylinder in a longitudinal view and shows a more granular appearance in sperm cells from the seminal vesicle in comparison to a more electron-dense appearance in sperm cells from the seminal uterine receptacle.

Testicular structure and development of germ cells of Hypophthalmus marginatus Valenciennes 1840 (Siluriformes: Pimelodidae)

The present study was conducted to assess the testicular structure and germ cell ultrastructure of Hypophthalmus marginatus during spermatogenesis. Semen and sections of the mid-region of the testis were collected, processed, and analyzed using optical and electron microscopy. Macroscopically, the testes of H. marginatus were filiform, and the testicular parenchyma was composed of spermatogenic cells that proliferated, organized within spermatic cysts. During spermiogenesis, spermatids had no nuclear rotation. The proximal centriole was perpendicular to the distal centriole, characteristic of type III spermiogenesis. Spermatozoa were released into the lumen of the seminiferous tubules and had an ovoid head without an acrosome, condensed nucleus, and shallow nuclear fossa. The midpiece was short, with a single long flagellum. The flagellum had the typical axoneme structure, with nine pairs of peripheral and a central pair of microtubules. The thin end piece comprised only peripheral microtubules. Spermatogenesis in H. marginatus features filiform testes, cystic spermatogenesis, and type III spermiogenesis.

Ultrastructural feature of spermatogenic cells and spermatozoon in cultured burbot Lota lota

Testis development and ultrastructure of spermatogenic cells and spermatozoa of burbot Lota lota, a commercially important cold freshwater fish, were studied by light and transmission electron microscopy. Spermatogonia, spermatocytes, spermatids, and spermatozoa are distributed along the seminiferous tubules. Electron-dense bodies appear in germ cells from primary spermatogonia to secondary spermatocytes. We identified three distinct stages of spermatid cell differentiation based on chromatin condensation, development of the flagellum, formation of a nuclear fossa, and elimination of excess cytoplasm. Spermatozoa were anacrosomal and characterized by location of the centrioles outside the nuclear fossa and incomplete perpendicular arrangement of the centrioles. The sperm flagellum displayed an axoneme with nine doublets of peripheral microtubules and two central microtubules. These results provide valuable information for burbot taxonomy and may clarify the process of spermatogenesis for this species.