Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Summary

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Abstract

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Introduction

Skeletal muscle is the largest tissue in the human body, totaling 35%-40% of body mass¹. Satellite cells are muscle stem cells, anatomically characterized by their position (juxtaposed to the plasma membrane, underneath the basal lamina of muscle fibers), that give rise to proliferating myoblasts (myogenic progenitor cells), which eventually differentiate and integrate into existing myofibers and/or fuse to form new myofibers^2,3,4. Their discovery and the progress in the study of their biology has led to significant insights into muscle development and....

Protocol

All procedures involving animal subjects were approved by the San Raffaele Institutional Animal Care and Use Committee.

1. Dissection of mouse hindlimb muscles

1. Sterilize tweezers and scissors (both straight and curved) by autoclaving.
2. Prepare and filter (0.22 µm) all the media (blocking medium, digestion medium, proliferating medium, and differentiating medium) before starting the experiment (see Table of Materials).
3. Put 5 mL of phosphate-buffered saline (PBS) in a 35 mm Petri dish for muscle collection.
4. Sacrifice the mouse by cervical dislocation or by using CO₂ and....

Results

To automatically follow nuclear movement during myoblast differentiation in live imaging, the nuclei should preferentially be fluorescently labeled. It is important to note that using DNA-intercalating molecules is not feasible because these molecules interfere with the proliferation and differentiation of primary myoblasts¹³. As an example, proliferation and differentiation have been analyzed in primary myoblasts cultured with or without Hoechst (

Discussion

Muscle fibers (myofibers) are multinucleated cells that are formed by the fusion of muscle precursors cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation^2,3,4. To assess myoblast differentiation, the common procedure consists of culturing myoblasts in differentiating medium and fixing the cells at different time points to perform immunofluorescence staining for MyHC, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
chicken embryos extract	Seralab	CE-650-J
collagenase	Sigma	C9263-1G	125units/mg
collagen from calf skin	Sigma	C8919
dispase	Gibco	17105-041	1.78 units/mg
doxyciclin	Sigma	D1515
DMEM	Sigma	D5671
fetal bovine serum	Life technologies	10270106
gentamicin	Sigma	G1397
Horse serum	Invitrogen	16050-098
Hoechst	life technologies	33342
IMDM	Sigma	I3390
L-glutamine	Sigma	G7513
Matrigel	Corning	356231
penicillin-streptomycin	Sigma	P0781
red blood cells lysis medium	Biolegend	420301
Digestion medium
collagenase	40 mg
dispase	70 mg
PBS	20 ml
filtered 0.22um
Blocking medium
DMEM
Fetal bovine serum	10%
L-glutammine	1%
penicillin-streptomycin	1%
gentamicin	1 ‰
filtered 0.22um
proliferation medium
IMDM
Fetal bovine serum	20%
L-glutammine	1%
penicillin-streptomycin	1%
gentamicin	1 ‰
chichen embryo extract	3%
filtered 0.22um
differentiation medium
IMDM
Horse serum	2%
L-glutammine	1%
penicillin-streptomycin	1%
gentamicin	1 ‰
chichen embryo extract	1%
filtered 0.22um