scholarly journals Axonemal doublet microtubules can split into two complete singlets in human sperm flagellum tips

2019 ◽  
Author(s):  
Davide Zabeo ◽  
Jacob T Croft ◽  
Johanna L Höög
Keyword(s):  
Embryonic Development ◽  
Electron Tomography ◽  
Human Sperm ◽  
Human Spermatozoa ◽  
Model Organisms ◽  
Sperm Flagellum ◽  
Human Fertility ◽  
Cryo Electron Tomography ◽  
Health And Disease

AbstractMotile flagella are crucial for human fertility and embryonic development. The distal tip of the flagellum is where growth and intra-flagellar transport are coordinated. In most, but not all, model organisms the distal tip includes a “singlet region”, where axonemal doublet microtubules terminate and only complete A-tubules extend as singlet microtubules to the tip. How a human flagellar tip is structured is unknown. Here, the flagellar tip structure of human spermatozoa was investigated by cryo-electron tomography, revealing the formation of two complete singlet microtubules from both the A-tubule and B-tubule of doublet microtubules. This different tip arrangement in human spermatozoa shows the need to investigate human flagella directly in order to understand their role in our health and disease.

scholarly journals 3D structure and in situ arrangements of CatSper channel in the sperm flagellum

2021 ◽  
Author(s):  
Yanhe Zhao ◽  
Huafeng Wang ◽  
Caroline Wiesehoefer ◽  
Naman B Shah ◽  
Evan Reetz ◽  
...  
Keyword(s):  
Electron Tomography ◽  
3D Structure ◽  
Human Sperm ◽  
Structural Basis ◽  
Sperm Flagellum ◽  
Mammalian Sperm ◽  
Zigzag Pattern ◽  
Associated Proteins

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, the complex subunit composition of CatSper has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.

scholarly journals A lumenal interrupted helix in human sperm tail microtubules

2016 ◽  
Author(s):  
Davide Zabeo ◽  
John M. Heumann ◽  
Cindi L. Schwartz ◽  
Azusa Suzuki-Shinjo ◽  
Garry Morgan ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Human Sperm ◽  
Variable Number ◽  
Human Diseases ◽  
Human Spermatozoon ◽  
Sperm Tail ◽  
Swimming Direction ◽  
Left Handed ◽  
Cryo Electron Tomography ◽  
Cellular Extensions

AbstractEukaryotic flagella are complex cellular extensions involved in many human diseases gathered under the term ciliopathies. Currently, detailed insights on flagellar structure come from studies on protozoa. Here, cryo-electron tomography (cryo-ET) of intact human spermatozoon tails showed a variable number of microtubules in the singlet region. Inside their lumen, a novel left-handed interrupted helix which extends several micrometers at their plus ends was discovered. This structure was named Tail Axoneme Intra-Lumenal Spiral (TAILS) and binds directly to 11 protofilaments on the internal microtubule wall, coaxial with the surrounding microtubule lattice. It leaves a gap over the microtubule seam, which was directly visualized in both singlet and doublet microtubules. We suggest that TAILS may stabilize microtubules, enable rapid swimming or play a role in controlling the swimming direction of spermatozoa.

scholarly journals Fully automated, sequential focused ion beam milling for cryo-electron tomography

2019 ◽  
Author(s):  
Tobias Zachs ◽  
João M. Medeiros ◽  
Andreas Schertel ◽  
Gregor L. Weiss ◽  
Jannik Hugener ◽  
...  
Keyword(s):  
Cell Biology ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Model Organisms ◽  
User Input ◽  
Cryo Electron Tomography ◽  
Cellular Context ◽  
Rough Milling ◽  
Time Required

AbstractCryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, with the unique ability to determine structures of macromolecular complexes in their cellular context. A major limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

scholarly journals Fully automated, sequential focused ion beam milling for cryo-electron tomography

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Tobias Zachs ◽  
Andreas Schertel ◽  
João Medeiros ◽  
Gregor L Weiss ◽  
Jannik Hugener ◽  
...  
Keyword(s):  
Cell Biology ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Model Organisms ◽  
User Input ◽  
Cryo Electron Tomography ◽  
Cellular Context ◽  
Rough Milling ◽  
Time Required

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

scholarly journals Ciliary Proteins: Filling the Gaps. Recent Advances in Deciphering the Protein Composition of Motile Ciliary Complexes

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 730 ◽  
Author(s):  
Anna Osinka ◽  
Martyna Poprzeczko ◽  
Magdalena M. Zielinska ◽  
Hanna Fabczak ◽  
Ewa Joachimiak ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Higher Plants ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Protein Composition ◽  
Model Organisms ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Biochemical Analyses ◽  
Eukaryotic Organisms

Cilia are highly evolutionarily conserved, microtubule-based cell protrusions present in eukaryotic organisms from protists to humans, with the exception of fungi and higher plants. Cilia can be broadly divided into non-motile sensory cilia, called primary cilia, and motile cilia, which are locomotory organelles. The skeleton (axoneme) of primary cilia is formed by nine outer doublet microtubules distributed on the cilium circumference. In contrast, the skeleton of motile cilia is more complex: in addition to outer doublets, it is composed of two central microtubules and several diverse multi-protein complexes that are distributed periodically along both types of microtubules. For many years, researchers have endeavored to fully characterize the protein composition of ciliary macro-complexes and the molecular basis of signal transduction between these complexes. Genetic and biochemical analyses have suggested that several hundreds of proteins could be involved in the assembly and function of motile cilia. Within the last several years, the combined efforts of researchers using cryo-electron tomography, genetic and biochemical approaches, and diverse model organisms have significantly advanced our knowledge of the ciliary structure and protein composition. Here, we summarize the recent progress in the identification of the subunits of ciliary complexes, their precise intraciliary localization determined by cryo-electron tomography data, and the role of newly identified proteins in cilia.

A High Resolution Cytochemical Study of Nuclear ATPase in Human Spermatozoa

1975 ◽  
Vol 33 ◽  
pp. 594-595
Author(s):  
A. Sosa ◽  
L. Calzada
Keyword(s):  
High Resolution ◽  
Atpase Activity ◽  
Nuclear Membrane ◽  
Human Sperm ◽  
Sperm Nucleus ◽  
Human Spermatozoa ◽  
Cytochemical Study ◽  
Membrane Bound ◽  
Sperm Nuclei ◽  
Interchromatin Space

The dependence of nuclear metabolism on the function of the nuclear membrane is not well understood. Whether or not the function of the nuclear membrane is partial or totally responsible of the repressed template activity of human sperm nucleus has not at present been elucidated. One of the membrane-bound enzymatic activities which is concerned with the mechanisms whereby substances are thought to cross cell membranes is adenosintriphosphatase (ATPase). This prompted its characterization and distribution by high resolution photogrammetry on isolated human sperm nuclei. Isolated human spermatozoa nuclei were obtained as previously described. ATPase activity was demonstrated by the method of Wachstein and Meisel modified by Marchesi and Palade. ATPase activity was identified as dense and irregularly distributed granules confined to the internal leaflet of the nuclear membrane. Within the nucleus the appearance of the reaction product occurs as homogenous and dense precipitates in the interchromatin space.

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