Myofibers drive postnatal myonuclear accretion through Arp2/3-dependent membrane protrusions

This study demonstrates that the Arp2/3 complex in postnatal myofibers actively drives muscle growth by generating membrane protrusions that facilitate the fusion of satellite cell-derived myoblasts, thereby establishing myofibers as autonomous regulators of their own nuclear accretion.

The Gist

The Big Picture: Who is the Boss of Muscle Growth?

Imagine your body's muscles are like a massive construction site. For a muscle to grow bigger and stronger after you are born, it needs to add more "workers" (nuclei) to its team. These workers come from a pool of reserve laborers called Satellite Cells.

For a long time, scientists thought the story was simple: The reserve workers (Satellite Cells) were the active ones. They would run over to the muscle, knock on the door, and fuse themselves into the team to help it grow. The muscle itself was seen as a passive building, just waiting to be added to.

This paper flips the script. It discovers that the muscle isn't just a passive building; it's an active recruiter. The muscle itself reaches out, grabs the workers, and pulls them in.

The Main Character: The "Arp2/3" Tool Kit

To understand how this works, we need to meet the Arp2/3 complex. Think of this as a specialized construction tool kit found inside every cell. Its main job is to build "scaffolding" made of actin (a type of protein). This scaffolding allows cells to push their membranes out, creating little arms or fingers called protrusions.

Usually, we know this tool kit helps the workers (Satellite Cells) reach out and grab the muscle. But this study asked a big question: Does the muscle use this tool kit to reach back?

The Experiment: Turning Off the Tool Kit

The researchers created a special group of baby mice where they could turn off the "Arp2/3 tool kit" only inside the muscle cells, leaving the rest of the body (and the reserve workers) untouched.

What happened?

1. The Mice Got Weak: The baby mice couldn't walk straight. They dragged their feet, wobbled, and had trouble lifting their bodies. Their muscles were smaller and weaker than normal.
2. The Workers Were Stuck: The reserve workers (Satellite Cells) were fully awake and ready to work. In fact, they were so eager they were piling up outside the muscle doors, knocking frantically. But they couldn't get in.
3. The Muscle Didn't Reach Out: Without the tool kit, the muscle cells lost their ability to grow "arms." They couldn't reach out to grab the workers.

The "Aha!" Moment: The Muscle Reaches Out

The team then watched muscle cells and workers interacting under a microscope in a petri dish. Here is what they saw:

• The Dance: When a muscle cell met a worker, the muscle didn't just sit there. It grew long, living "fingers" (membrane protrusions) made of the Arp2/3 scaffolding.
• The Grab: These fingers wrapped around the worker, pulling them close until the two cells merged into one.
• The Proof: The researchers used a "remote control" (optogenetics) to force the muscle cells to grow these fingers even when they weren't supposed to. Result? The muscle cells immediately started grabbing and fusing with workers, even without any other signal. This proved that the muscle's ability to reach out is the key to the whole process.

The Analogy: The "Velcro" vs. The "Handshake"

• Old View: Imagine the Satellite Cell is a person with Velcro on their back, and the Muscle is a wall. The person runs and sticks to the wall.
• New View: Imagine the Muscle is a person with a long, sticky hand (the Arp2/3 protrusion). The Muscle reaches out, grabs the Satellite Cell, and pulls them in for a handshake (fusion). If the Muscle loses its hand (by removing Arp2/3), the Satellite Cell can run up to the wall all day, but no one is there to shake their hand, so they can't join the team.

Why This Matters

This discovery changes how we think about muscle growth and disease.

1. Muscles are Active: Muscles aren't just passive structures; they actively drive their own growth by reaching out to their stem cells.
2. New Hope for Disease: Some muscle diseases (like centronuclear myopathies) involve weak muscles and small muscle fibers. This research suggests that if we can figure out how to fix the "tool kit" (Arp2/3) inside the muscle, we might be able to help muscles grow back stronger, even if the reserve workers are healthy.

In short: The muscle isn't just waiting to be built; it's the one holding the hammer and reaching out to build itself.

Technical Summary

1. Problem Statement

Skeletal muscle growth during postnatal development relies on the fusion of satellite cell (SC)-derived myoblasts with pre-existing myofibers to increase myofiber size and nuclear number (myonuclear accretion). While the role of the actin nucleator Arp2/3 complex in driving invasive protrusions for myoblast fusion during embryogenesis is well-established, its role within the mature myofiber during postnatal growth remains unknown. It is unclear whether myofibers are passive recipients of fusion or active participants that regulate the process.

2. Methodology

The authors employed a multi-faceted approach combining in vivo mouse models, in vitro co-culture systems, live imaging, and optogenetics:

• Genetic Models:
  • Generated a skeletal muscle-specific, inducible Arpc4 knockout (Arpc4-/-) mouse model using the HSA-MCM system (Cre-ERT2 under the human $\alpha$-actin promoter). Tamoxifen was administered at postnatal days (P) 1–3 to deplete Arpc4 specifically in myofibers.
  • Created control lines and specific knockouts for Arpc5 and Arpc5l isoforms to test for isoform dependency.
• Phenotypic Analysis:
  • Assessed motor function in neonates (P7) using open-field tests (ambulation scores, foot angles) and hindlimb suspension tests.
  • Performed histological analysis (H&E, immunostaining for laminin, PCM-1, Pax7, Ki-67, MyoD) to evaluate myofiber cross-sectional area (CSA), nuclear positioning, and satellite cell activation status.
  • Used EdU labeling in vivo to distinguish newly fused nuclei (EdU+) from pre-existing myonuclei.
• In Vitro Co-culture & Live Imaging:
  • Developed a co-culture system where FACS-sorted SCs were differentiated into myofibers (D3) and co-cultured with mononucleated myoblasts.
  • Used live-cell imaging (tdTomato-myoblasts + CAAX-GFP-myofibers) to visualize membrane dynamics.
  • Tracked Arp2-mNG localization to determine Arp2/3 accumulation at contact sites.
• Optogenetics:
  • Employed a Cdc42 optogenetic tool (mCherry-ITSN-CRY2) to induce Arp2/3-dependent membrane protrusions in myofibers via 488 nm light activation, testing if protrusion alone is sufficient to drive fusion.
• Electron Microscopy:
  • Used Transmission Electron Microscopy (TEM) on P7 muscle sections to visualize ultrastructural membrane protrusions between myofibers and myoblasts.

3. Key Contributions

• Repositioning the Myofiber: The study challenges the dogma that myoblasts are the sole drivers of fusion. It demonstrates that myofibers are active, fusogenic partners that actively extend membrane protrusions to capture and fuse with myoblasts.
• Mechanism of Action: Identifies a specific Arp2/3-dependent mechanism within the myofiber cytoskeleton that generates long-lived membrane protrusions essential for postnatal nuclear accretion.
• Isoform Independence: Establishes that this postnatal fusion mechanism is distinct from the Arpc5-isoform-dependent mechanisms previously shown to govern myonuclear migration, indicating a robust, isoform-independent fusion machinery.

4. Key Results

• In Vivo Phenotype:

  • Muscle Weakness: Arpc4-/- mice exhibited significant motor deficits, including increased immobility, asymmetric gait, wider foot angles (splayed paws), and reduced hindlimb strength (fewer pulls in suspension tests).
  • Reduced Growth: Myofibers in Arpc4-/- mice had a significantly smaller cross-sectional area (CSA) and fewer myonuclei compared to controls.
  • Accumulation of SCs: Despite normal SC activation (Pax7+/Ki-67+) and differentiation (MyoD+), activated SCs failed to fuse. They accumulated in clusters around Arpc4-/- myofibers, indicating a myofiber-intrinsic defect in fusion rather than a lack of donor cells.
  • EdU Labeling: In vivo EdU incorporation revealed a drastic reduction in new nuclear accretion in Arpc4-/- myofibers, confirming impaired fusion.

• In Vitro Mechanism:

  • Protrusion Formation: Live imaging showed that wild-type (WT) myofibers form long-lived membrane protrusions (avg. 30 min, some >2h) enriched with Arp2/3 (Arp2-mNG) at sites of contact with myoblasts.
  • Arp2/3 Requirement: Depletion of Arpc4 in myofibers (via siRNA or Cre) reduced the number of protrusions by ~3-fold and significantly decreased the Myonuclear Accretion Index (MAI).
  • Optogenetic Sufficiency: Optogenetic activation of Cdc42 in myofibers (inducing protrusions) was sufficient to induce fusion with adjacent myoblasts (50% fusion rate), even without other stimuli, proving that protrusive activity drives the fusion event.

• Ultrastructural Evidence:

  • TEM analysis confirmed that ~80% of WT myofiber-myoblast interactions involved membrane protrusions, whereas 60% of Arpc4-/- interactions lacked protrusions entirely. The few protrusions in mutants were significantly smaller.

• Isoform Specificity:

  • Depletion of Arpc5 or Arpc5l isoforms did not affect myofiber size or nuclear number, confirming that the postnatal fusion defect in Arpc4-/- mice is not due to the loss of specific Arpc5 isoforms but rather the core Arp2/3 function.

5. Significance

• Paradigm Shift: This work fundamentally shifts the understanding of muscle growth, identifying the mature myofiber as an active regulator of its own nuclear accretion via Arp2/3-dependent protrusions.
• Therapeutic Implications: The findings suggest that defects in Arp2/3-mediated myofiber protrusions could underlie congenital myopathies characterized by small myofibers and weakness (e.g., centronuclear myopathies).
• Cell-Cell Fusion Mechanism: It provides a novel mechanism for cell-cell fusion where the "receiving" cell actively extends cytoskeletal structures to facilitate membrane juxtaposition and fusion, a concept potentially applicable to other fusion events in development and regeneration.

In conclusion, the paper demonstrates that the Arp2/3 complex in myofibers is essential for generating membrane protrusions that actively drive myoblast fusion, thereby enabling postnatal muscle growth and strength. Loss of this mechanism leads to a failure in nuclear accretion and muscle weakness.