The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure.

Conception in mammals is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum can be divided into the midpiece, the principal piece and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility; however, the actin organization in sperm remains elusive. In the present work, we show that there are different types of actin structures in the sperm tail by using three-dimensional stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell.

Growth Progression of Oxygenic Photogranules and Its Impact on Bioactivity for Aeration-Free Wastewater Treatment.

Oxygenic photogranules (OPGs), spherical aggregates comprised of phototrophic and nonphototrophic microorganisms, treat wastewater without aeration, which currently incurs the highest energy demand in wastewater treatment. In wastewater-treatment reactors, photogranules grow in number as well as in size. Currently, it is unknown how the photogranules grow in size and how the growth impacts their properties and performance in wastewater treatment. Here, we present that the photogranules' growth occurs with changes in phototrophic community and granular morphology. We observed that as the photogranules grow larger, filamentous cyanobacteria become enriched while other phototrophic microbes diminish significantly. The photogranules greater than 3 mm in diameter showed the development of a layered structure in which a concentric filamentous cyanobacterial layer encloses noncyanobacterial aggregates. We observed that the growth of photogranules significantly impacts their capability of producing oxygen, the key element in OPG wastewater treatment. Among seven size classes investigated in this study, photogranules in the 0.5-1 mm size group showed the highest specific oxygen production rate (SOPR), 21.9 ± 1.3 mg O2/g VSS-h, approximately 75% greater than the SOPR of mixed photogranular biomass. We discuss engineering the OPG process based on photogranules' size, promoting the stability of the granular process and enhancing efficiency for self-aerating wastewater treatment.

Survival of Polyploid hybrid salamander embryos.

BACKGROUND: Animals with polyploid, hybrid nuclei offer a challenge for models of gene expression and regulation during embryogenesis. To understand how such organisms proceed through development, we examined the timing and prevalence of mortality among embryos of unisexual salamanders in the genus Ambystoma. RESULTS: Our regional field surveys suggested that heightened rates of embryo mortality among unisexual salamanders begin in the earliest stages of embryogenesis. Although we expected elevated mortality after zygotic genome activation in the blastula stage, this is not what we found among embryos which we reared in the laboratory. Once embryos entered the first cleavage stage, we found no difference in mortality rates between unisexual salamanders and their bisexual hosts. Our results are consistent with previous studies showing high rates of unisexual mortality, but counter to reports that heightened embryo mortality continues throughout embryo development. CONCLUSIONS: Possible causes of embryonic mortality in early embryogenesis suggested by our results include abnormal maternal loading of RNA during meiosis and barriers to insemination. The surprising survival rates of embryos post-cleavage invites further study of how genes are regulated during development in such polyploid hybrid organisms.

Investigation of the Fate and Dynamics of Extracellular Polymeric Substances (EPS) during Sludge-Based Photogranulation under Hydrostatic Conditions.

Oxygenic photogranules have received increasing interest due to their ability to treat wastewater without aeration and recover wastewater's chemical energy and solar energy. It has been reported that these photogranules can be produced under both hydrostatic and hydrodynamic conditions, and enrichment of filamentous cyanobacteria is required for this photogranulation to occur. Despite the critical role extracellular polymeric substances (EPS) play in granulation, EPS in photogranulation is yet virtually unknown. Here, we present the fate and dynamics of different fractions of EPS in sludge-based photogranulation under hydrostatic conditions. The study shows that during the transformation of activated sludge into a photogranular biomass, sludge's base-extractable proteins selectively degrade. Strong correlations between base-extracted proteins and the growth of chlorophyll a and chlorophyll a/ b ratio suggest that the bioavailability of this organic nitrogen is linked with selection and enrichment of filamentous cyanobacteria under hydrostatic conditions. The results of soluble and sonication-extractable EPS and microscopy also show that the growth of filamentous cyanobacteria required large amounts of polysaccharide-based EPS for their motility and maintenance. With findings on the progression of photogranulation, the fate and dynamics of EPS, and microscopy on microstructures associated with EPS, we discuss potential mechanisms of photogranulation occurring under hydrostatic conditions.

The importance of filamentous cyanobacteria in the development of oxygenic photogranules.

Microorganisms often respond to their environment by growing as densely packed communities in biofilms, flocs or granules. One major advantage of life in these aggregates is the retention of its community in an ecosystem despite flowing water. We describe here a novel type of granule dominated by filamentous and motile cyanobacteria of the order Oscillatoriales. These bacteria form a mat-like photoactive outer layer around an otherwise unconsolidated core. The spatial organization of the phototrophic layer resembles microbial mats growing on sediments but is spherical. We describe the production of these oxygenic photogranules under static batch conditions, as well as in turbulently mixed bioreactors. Photogranulation defies typically postulated requirements for granulation in biotechnology, i.e., the need for hydrodynamic shear and selective washout. Photogranulation as described here is a robust phenomenon with respect to inoculum characteristics and environmental parameters like carbon sources. A bioprocess using oxygenic photogranules is an attractive candidate for energy-positive wastewater treatment as it biologically couples CO2 and O2 fluxes. As a result, the external supply of oxygen may become obsolete and otherwise released CO2 is fixed by photosynthesis for the production of an organic-rich biofeedstock as a renewable energy source.

Conserved structural motifs in the central pair complex of eukaryotic flagella.

Cilia and flagella are conserved hair-like appendages of eukaryotic cells that function as sensing and motility generating organelles. Motility is driven by thousands of axonemal dyneins that require precise regulation. One essential motility regulator is the central pair complex (CPC) and many CPC defects cause paralysis of cilia/flagella. Several human diseases, such as immotile cilia syndrome, show CPC abnormalities, but little is known about the detailed three-dimensional (3D) structure and function of the CPC. The CPC is located in the center of typical [9+2] cilia/flagella and is composed of two singlet microtubules (MTs), each with a set of associated projections that extend toward the surrounding nine doublet MTs. Using cryo-electron tomography coupled with subtomogram averaging, we visualized and compared the 3D structures of the CPC in both the green alga Chlamydomonas and the sea urchin Strongylocentrotus at the highest resolution published to date. Despite the evolutionary distance between these species, their CPCs exhibit remarkable structural conservation. We identified several new projections, including those that form the elusive sheath, and show that the bridge has a more complex architecture than previously thought. Organism-specific differences include the presence of MT inner proteins in Chlamydomonas, but not Strongylocentrotus, and different overall outlines of the highly connected projection network, which forms a round-shaped cylinder in algae, but is more oval in sea urchin. These differences could be adaptations to the mechanical requirements of the rotating CPC in Chlamydomonas, compared to the Strongylocentrotus CPC which has a fixed orientation.

The structural heterogeneity of radial spokes in cilia and flagella is conserved.

Radial spokes (RSs) are ubiquitous components of motile cilia and flagella and play an essential role in transmitting signals that regulate the activity of the dynein motors, and thus ciliary and flagellar motility. In some organisms, the 96 nm axonemal repeat unit contains only a pair of spokes, RS1 and RS2, while most organisms have spoke triplets with an additional spoke RS3. The spoke pairs in Chlamydomonas flagella have been well characterized, while spoke triplets have received less attention. Here, we used cryoelectron tomography and subtomogram averaging to visualize the three-dimensional structure of spoke triplets in Strongylocentrotus purpuratus (sea urchin) sperm flagella in unprecedented detail. Only small differences were observed between RS1 and RS2, but the structure of RS3 was surprisingly unique and structurally different from the other two spokes. We observed novel doublet specific features that connect RS2, RS3, and the nexin-dynein regulatory complex, three key ciliary and flagellar structures. The distribution of these doublet specific structures suggests that they could be important for establishing the asymmetry of dynein activity required for the oscillatory movement of cilia and flagella. Surprisingly, a comparison with other organisms demonstrated both that this considerable RS heterogeneity is conserved and that organisms with RS pairs contain the basal part of RS3. This conserved RS heterogeneity may also reflect functional differences between the spokes and their involvement in regulating ciliary and flagellar motility.

Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella.

Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo-electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named "radial spoke 3 stand-in," which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.