Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Actin-myosin interactions visualized by the quick-freeze, deep-etch replica technique.

J E Heuser, R Cooke

    Journal of Molecular Biology
    |September 5, 1983
    PubMed
    Summary

    A new quick-freeze, deep-etch electron microscopy method reveals actin filament structure and insect muscle crossbridges. Sample preparation significantly impacts observed crossbridge shape and orientation, complicating visualization of natural muscle movements.

    Related Concept Videos

    You might also read

    Related Articles

    Articles linked to this work by shared authors, journal, and citation graph.

    Sort by
    Same author

    The Purdue Pegboard test for surveillance of vibration-exposed workers.

    Occupational medicine (Oxford, England)·2025
    Same author

    Cosmology and fundamental physics with the ELT-ANDES spectrograph.

    Experimental astronomy·2024
    Same author

    Early-life gut bacterial community structure predicts disease risk and athletic performance in horses bred for racing.

    Scientific reports·2024
    Same author

    COVID-19 associated with Raynaud's phenomenon in a vibration-exposed worker.

    Occupational medicine (Oxford, England)·2023
    Same author

    Successful rehabilitation and release of a powerful owl chick with suspected rodenticide poisoning.

    Australian veterinary journal·2023
    Same author

    Behavioural intervention to promote the uptake of planned care in urgent dental care attenders: study protocol for the RETURN randomised controlled trial.

    Trials·2022

    Area of Science:

    • Structural biology
    • Biophysics
    • Cell biology

    Background:

    • Understanding the precise structure of cytoskeletal components like actin filaments and muscle crossbridges is crucial for elucidating cellular mechanics.
    • Traditional electron microscopy techniques, such as negative staining, can introduce artifacts that may obscure fine structural details or lead to misinterpretations.

    Purpose of the Study:

    • To investigate the ultrastructure of actin filaments, acto-myosin interactions, and insect flight muscle crossbridges using a novel quick-freeze, deep-etch electron microscopy technique.
    • To compare the structural information obtained from this new method with that from conventional techniques like negative staining.
    • To assess the influence of different sample preparation protocols on the observed morphology and orientation of muscle crossbridges.

    Main Methods:

    Related Experiment Videos

    • Biological samples were prepared using a quick-freeze, deep-etch, rotary-platinum replication, and transmission electron microscopy approach.
    • Actin filaments and actin filaments decorated with myosin subfragment-1 (S1) were examined.
    • Insect flight muscles in rigor and relaxed states were analyzed, with variations in fixation and staining protocols.

    Main Results:

    • Quick-freeze, deep-etch revealed actin filaments with prominent transverse bands consistent with a left-handed helix, though the two-start helix was less pronounced than in negatively stained samples.
    • Acto-S1 complexes showed the outer envelope of S1 but lacked the distinct "arrowheads" seen with negative staining, appearing only slightly polarized.
    • Insect flight muscle crossbridges were clearly visualized, appearing numerous and regularly arranged in rigor muscles and sparse in relaxed muscles.
    • Crossbridge orientation in insect muscles varied significantly with preparation methods, from approximately 80 degrees in aldehyde-fixed samples to 45 degrees after tannic acid treatment, and could be stretched in unfixed samples.

    Conclusions:

    • The quick-freeze, deep-etch method provides high-resolution structural details of actin filaments and muscle crossbridges.
    • Observed differences in actin filament structure and acto-S1 appearance between freeze-etching and negative staining highlight the impact of preparation techniques.
    • Crossbridge shape and orientation are highly sensitive to sample preparation methods, posing challenges for accurately visualizing natural muscle movements via electron microscopy.