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

Cellular localization of activated N-WASP using a conformation-sensitive antibody.

P Sukumvanich1, V DesMarais, C V Sarmiento

  • 1Department of Obstetrics, Gynecology, and Women's Health, Division of Gynecologic Oncology, Albert Einstein College of Medicine, Bronx, NY, USA.

Cell Motility and the Cytoskeleton
|September 14, 2004
PubMed
Summary

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 role of adjuvant treatment for early-stage uterine clear cell carcinomas.

Gynecologic oncology·2023
Same author

Is Substantial Lymphovascular Space Invasion Prognostic for Clinical Outcomes in Type II Endometrial Cancer?

Clinical oncology (Royal College of Radiologists (Great Britain))·2022
Same author

Cost-effectiveness analysis of tumor molecular classification in high-risk early-stage endometrial cancer.

Gynecologic oncology·2021
Same author

Early outcomes after definitive chemoradiation therapy with Vienna/Venezia hybrid high-dose rate brachytherapy applicators for cervical cancer: A single-institution experience.

Brachytherapy·2020
Same author

Regional Control and Chemoradiotherapy Dose Response for Clinically Involved Lymph Nodes in Patients with Locally Advanced Endometrial Cancers Who are Not Candidates for Upfront Surgical Staging Extrafascial Hysterectomy.

Clinical oncology (Royal College of Radiologists (Great Britain))·2020
Same author

Is the risk of substantial LVSI in stage I endometrial cancer similar to PORTEC in the North American population? - A single-institution study.

Gynecologic oncology·2020

Epidermal Growth Factor (EGF) stimulation activates N-WASP at the cell's leading edge, revealing its role in lamellipod formation. This study introduces a novel method to track protein activation dynamics.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The Arp2/3 complex is crucial for actin nucleation, with N-WASP and Scar/WAVE proteins as key regulators.
  • Understanding the spatiotemporal regulation of N-WASP activation is essential for deciphering cell motility and morphology.
  • Carcinoma cell dynamics are influenced by actin cytoskeleton rearrangements, particularly at the leading edge.

Purpose of the Study:

  • To investigate the recruitment and activation dynamics of N-WASP in carcinoma cells following EGF stimulation.
  • To develop and validate a conformation-sensitive antibody (CSA) for detecting active N-WASP.
  • To determine the localization of activated N-WASP within the cell during dynamic processes.

Main Methods:

  • Utilized EGF stimulation in carcinoma cells to induce dynamic cellular responses.

Related Experiment Videos

  • Developed a conformation-sensitive antibody (CSA) targeting the CRIB domain of N-WASP to distinguish active from inactive states.
  • Performed in vitro experiments including immunoprecipitation and Cdc42 activation assays to validate CSA specificity.
  • Conducted cell staining experiments to assess N-WASP accessibility to CSA and its localization.

    • Main Results:

    • N-WASP is recruited to the leading edge nucleation zone within 1 minute of EGF stimulation, mirroring barbed-end formation.
    • The conformation-sensitive antibody (CSA) specifically recognized active N-WASP, validated through in vitro assays.
    • Activated N-WASP was detected at the leading edge of lamellipods approximately 40 seconds after EGF stimulation.
    • The localization of activated N-WASP in lamellipods was an unexpected finding, contrasting with its known role in filopodia.

    Conclusions:

    • Active N-WASP is present in the nucleation zone of lamellipods, suggesting a role beyond filopodia formation.
    • The study demonstrates the feasibility of using conformation-specific antibodies to track protein activation status and localization in real-time.
    • This approach provides a novel window into the dynamic regulation of cellular processes involving N-WASP.