Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging

Chinedu U. Nworu, Paul A Krieg, Carol Gregorio

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Mutations in several sarcomeric proteins have been linked to various human myopathies. Therefore, having an in vivo developmental model available that develops quickly and efficiently is key for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; this is the pivotal foundation for deciphering disease mechanisms as well as the development of myopathy-related therapeutics. Although striated muscle cell culture studies have been extremely informative in providing clues to both the distribution and functions of sarcomeric proteins, myocytes in vivo develop in an irreproducible 3D environment. Xenopus laevis (frog) embryos are cost effective, compliant to protein level manipulations and develop relatively quickly (≤ a week) in a petri dish, thus providing a powerful system for de novo myofibrillogenesis studies. Although fluorophore-conjugated phalloidin labeling is the gold standard approach for investigating actin-thin filament architecture, it is well documented that phalloidin-labeling can be challenging and inconsistent within Xenopus embryos. Therefore we highlight several techniques that can be utilized to preserve both antibody and fluorophore-conjugated phalloidin labeling within Xenopus embryos for high-resolution fluorescence microscopy.

Original languageEnglish (US)
Pages (from-to)370-379
Number of pages10
JournalMethods
Volume66
Issue number3
DOIs
StatePublished - Apr 1 2014

Fingerprint

Xenopus Proteins
Phalloidine
Muscle Proteins
Labeling
Muscle
Embryonic Structures
Fluorophores
Muscular Diseases
Xenopus
Imaging techniques
Muscle Cells
Proteins
Striated Muscle
Muscle Development
Myofibrils
Fluorescence microscopy
Xenopus laevis
Actin Cytoskeleton
Fluorescence Microscopy
Cell culture

Keywords

  • Immunofluorescence microscopy
  • Myofibrillogenesis
  • Phalloidin
  • Sarcomere
  • Xenopus laevis

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging. / Nworu, Chinedu U.; Krieg, Paul A; Gregorio, Carol.

In: Methods, Vol. 66, No. 3, 01.04.2014, p. 370-379.

Research output: Contribution to journalArticle

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