Myo1e/f regulate phagocytic podosomes to promote efficient cup closure in macrophages

This study demonstrates that the class I myosins Myo1e and Myo1f are essential for coordinating the spatiotemporal transition from protrusive podosomes to a contractile phagocytic ring, thereby ensuring efficient cup closure and preventing stalled engulfment or excessive trogocytosis during Fc-receptor-mediated phagocytosis in macrophages.

The Gist

Imagine your body's immune system as a team of tiny, hungry security guards called macrophages. Their job is to spot invaders (like bacteria or debris) and swallow them whole. To do this, the guard has to stretch out its "arms" (made of a flexible material called actin) to wrap around the target and pull it inside. This process is called phagocytosis.

This new paper is about two specific tools these guards use to get the job done: tiny molecular motors called Myo1e and Myo1f. Think of these motors as the "glue" and "hands" that connect the guard's outer skin (the membrane) to its internal muscle fibers (the actin).

Here is what the researchers discovered, explained simply:

1. The Problem: Guards Without Tools

The scientists created a group of macrophages that were missing both Myo1e and Myo1f. It was like taking away the hands and glue from the security guards.

• The Result: These guards were terrible at their job. They tried to grab the targets but couldn't swallow them efficiently. They often got stuck halfway through, like someone trying to eat a giant burger but dropping it before taking a bite.

2. The Normal Process: A Well-Orchestrated Dance

When the guards do have these tools, the process is a smooth, three-step dance:

• Step 1: The Anchors (Podosomes): First, the guard sets up little suction-cup anchors on the ground to hold its position.
• Step 2: The Teeth (Actin Teeth): As the guard reaches for the target, it forms little "teeth" of muscle along the edge of the opening to grip the target tightly.
• Step 3: The Closing Ring: Finally, all those anchors and teeth rearrange into a strong, tight circle (a contractile ring) that squeezes the target inside.

3. What Went Wrong Without the Tools?

When the Myo1e/f motors were missing, the dance fell apart:

• No Anchors or Teeth: The guards couldn't set up their suction cups or form the gripping "teeth."
• Rushing the Finish: Because the early steps failed, the final "closing ring" formed way too early, before the target was actually inside.
• The Stuck Burger: This premature closing caused the process to stall. The guard would try to grab the target, fail, let go, and try again repeatedly, never successfully swallowing the whole thing.

4. The "Trogocytosis" Glitch

The paper also found a weird side effect. Instead of swallowing the whole target, the guards with missing tools started "nibbling" on it. They would take tiny, partial bites (a process called trogocytosis) rather than eating the whole meal. It's like trying to eat a whole apple but only managing to scrape off a few tiny pieces of skin.

5. The Secret Teamwork

The researchers looked closely at how the muscles were arranged and found a specific teamwork issue:

• Inside the Ring: Myo1e/f usually sit on the inside of the closing circle, acting as the hands pulling the skin inward.
• Outside the Ring: Another muscle, called Non-Muscle Myosin II, sits on the outside, acting like a belt to tighten the circle.
• The Mix-Up: Without Myo1e/f, the "belt" muscle (Myosin II) got confused and spread out everywhere instead of staying in a tight line. Without the inner hands to guide it, the outer belt couldn't do its job properly.

The Bottom Line

The paper concludes that Myo1e and Myo1f are essential coordinators. They make sure the guard builds the right anchors and teeth before trying to close the door. They balance the forces so the guard can smoothly transition from reaching out to pulling in, ensuring the target gets swallowed whole rather than just nibbled or dropped.

Technical Summary

Technical Summary: Myo1e/f Regulate Phagocytic Podosomes to Promote Efficient Cup Closure in Macrophages

1. Problem Statement

Phagocytosis is a critical immune function requiring the precise spatiotemporal coordination of the actin cytoskeleton to generate the protrusive and contractile forces necessary for target engulfment. While Class I myosins, specifically Myo1e and Myo1f (collectively Myo1e/f), are known to link the plasma membrane to the actin network, their specific mechanistic roles during Fc-receptor (FcR)-mediated phagocytosis have remained unclear. A key gap in understanding involves how these myosins regulate the dynamic transition from initial adhesion to the formation of a contractile phagocytic ring and the subsequent closure of the phagocytic cup.

2. Methodology

The study employed a multi-faceted experimental approach combining genetic manipulation, advanced imaging, and functional assays:

• Genetic Model: The researchers utilized CRISPR-Cas9 technology to generate RAW 264.7 macrophage cell lines lacking both Myo1e and Myo1f (double knockout, dKO).
• Rescue Experiments: To confirm specificity, dKO cells were re-expressed with either Myo1e or Myo1f to observe phenotypic restoration.
• Imaging Techniques:
  • Lattice-light-sheet microscopy (LLSM): Used for high-speed, high-resolution 3D imaging to capture dynamic actin remodeling without significant phototoxicity.
  • Confocal microscopy: Employed to visualize specific protein localizations and F-actin architectures.
• Functional Assays:
  • Quantification of IgG-coated bead uptake to measure phagocytic efficiency.
  • Analysis of trogocytosis (partial ingestion) using antibody-opsonized HL-60 cells.
• Protein Localization Analysis: Investigated the spatial distribution of Myo1e/f, F-actin, and non-muscle myosin II (NM2) during different stages of cup formation.

3. Key Contributions

This study provides the first comprehensive characterization of the dual role of Myo1e/f in macrophage phagocytosis, specifically defining their function in organizing phagocytic podosomes. The authors identified distinct actin architectures that drive cup progression and demonstrated that Myo1e/f are essential for the temporal and spatial coordination between protrusive forces (podosomes) and contractile forces (the phagocytic ring).

4. Key Results

• Phenotypic Defects in dKO Cells:
  • Myo1e/f-deficient macrophages exhibited a marked reduction in the uptake of IgG-coated beads.
  • This defect was partially rescued by re-introducing either Myo1e or Myo1f, confirming their functional redundancy and necessity.
• Disruption of Actin Architecture:
  • Wild-type (WT) cells displayed a sequential progression of actin structures: basal podosome-like adhesions $\rightarrow$ individual phagocytic podosomes (or "actin teeth") along the cup rim $\rightarrow$ reorganization into a higher-order contractile phagocytic ring.
  • dKO cells failed to form robust podosomes and lacked "actin teeth." Consequently, the phagocytic ring formed prematurely, leading to stalled cup progression and repeated, failed engulfment attempts.
• Spatial Regulation of Myosins:
  • Myo1e/f localized to both the podosomes and the inner surface of the phagocytic ring.
  • Non-muscle myosin II (NM2) localized to the outer surface of the ring.
  • In the absence of Myo1e/f, NM2 distribution became diffuse rather than organized, suggesting Myo1e/f are required to anchor or organize NM2 for effective contraction.
• Shift in Ingestion Mechanism:
  • dKO macrophages showed increased trogocytosis of antibody-opsonized HL-60 cells. This indicates a shift from efficient whole-target engulfment to partial target ingestion when the actin coordination machinery is compromised.

5. Significance

The findings establish that Myo1e and Myo1f are critical regulators of the force balance during phagocytosis. Their primary significance lies in:

1. Mechanistic Insight: They coordinate the transition from protrusive actin networks (podosomes) to contractile networks (the phagocytic ring), ensuring the cup progresses efficiently rather than stalling or closing prematurely.
2. Adhesion and Force Balance: Myo1e/f act as a molecular bridge that organizes NM2, ensuring that contractile forces are applied correctly to close the cup around the target.
3. Pathophysiological Implication: The shift toward trogocytosis in the absence of these myosins suggests that defects in Myo1e/f could compromise immune clearance efficiency, potentially leading to incomplete pathogen removal or altered immune signaling.

In conclusion, this paper redefines the role of Class I myosins in macrophages, moving beyond simple membrane-actin linking to a sophisticated regulatory role in the spatiotemporal orchestration of the phagocytic cup.