Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis

Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis

cite: Freeman, Spencer A., et al. “Integrins form an expanding diffusional barrier that coordinates phagocytosis.” Cell 164.1-2 (2016): 128-140.

Brief

To create the large zone of Src-family kinase (SFK) phosphorylation required for phagocytosis, a cascade of integrin activation, emanating from antigen contact sites, generates an expanding actin-based diffusion barrier that restricts the access of the bulky phosphatase molecules that target SFK.

Highlights

• Tyrosine phosphatases are excluded from sites of phagocytosis
• An expanding diffusion barrier prevents phosphatase access to sites of phagocytosis
• Integrins activated by phagocytic receptors generate the diffusion barrier
• Activated integrins bridge sparse phagocytic receptors and coordinate phagocytosis

Summary

© Spencer A Freeman, et al.

Phagocytosis activate

• Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs).
• Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement.

Fcg

• The mobility of CD45 increased markedly upon engagement of Fcg receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier.
  • By micropatterning IgG, the ligand of Fcg receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone.
  • Second messengers generated by Fcg receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcg receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

Introduction

• Phagocytosis is initiated by a clustering of receptors.

• Fcg receptor could recognize the Fc portion of IgG in phagocytic response.

• The Multiplicity of IgG stimulate the Src-family kinase (SFKs)

• CD45 and CD148 on phagocytes are tyrosine phosphatases.

• SFKs activation needs the remove of phosphatases.

• Upon binding ligand, B/T cell receptors to form a central supermolecular activation center (cSMAC)

• phosphatases are displaced to the periphery

• exofacial domain of Fcg receptors is notably shorter (z6 nm) than phosphatases. So, how they excluded CD45 and CD148?

• integrins as progressive diffusional barriers that serve to integrate the signals emanating from immobile Fcg receptor microclusters.

Result

Activation of Fcg Receptors Increases the Mobility of CD45

(A) Human macrophages incubated with polystyrene beads opsonized with 0.5 mg IgG/107 particles. Cells were fixed after 45 s and stained for CD45 (green), pY (red), and IgG (cyan, inset). Scale bar, 10 μm.

(B–F) Single CD45 particles were visualized in macrophages using anti-CD45 Fab fragments labeled with Qdots (B, inset).

(B) Macrophages were seeded onto either BSA-coated or IgG-coated coverslips and particles tracked for 20 s at 33 Hz. CD45 trajectories were analyzed by MSS; the motion type for each trajectory is color coded as follows: blue, confined; cyan, free; and red, linear. Scale bar, 5 μm.

(C–E) CD45 motion type ©, median confinement diameter (D), and median diffusion coefficient (E) for cells seeded on BSA (20 min), IgG (20 min), or BSA + LatA (15 + 5 min) were determined from 10-s recordings. Horizontal lines are means of ≥17 cells from three independent experiments; >1,000 trajectories were analyzed per condition.

(F) Three trajectories from (B) shown with color-coded time course. Depletion zones drawn in (B) and (F) delineate the area depleted of CD45. They were drawn to approximate the contour of the F-actin ring.

(A) shows that the CD45 was depleted from the phagocytic site. But the pY was recruited. As a result, they formed a phagocytic cup which had py in the center which was circled by CD45

(B) shows the migration of the CD45 during the phagocytosis.
Resting: When the cell didn’t perform phagocytosis
2/5/10 min: 2/5/10 min after the cell perform phagocytosis

• Contrary to the predictions made by the model of Yamauchi et al. (2012), fewer than 3% of the CD45 molecules moved linearly either at rest or during phagocytosis (Figures 1B and 1C).
  • Yamauchi et al. (2012) proposed that myosin II facilitates the redistribution of CD45 during phagocytosis.

© The different diffusion types of the CD45 contour with different particles.

engaging Fcg receptors increased the fraction of CD45 undergoing free diffusion from 47% ± 14% to 70% ± 11% (means ± SD) at the expense of confined molecules, which decreased proportionately

(D) the mean diameter of the remaining confinement zones increased.

CD45 Is Progressively Displaced by a Diffusion Barrier

MMS: diffusing freely
Video evidence: CD45 bounce off the edge of the zone of depletion

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IgG dots on the slice: printed IgG onto glass coverslips

The Area of Depletion of CD45 Extends beyond Regions of Microclustered Fcg Receptors

This is a fine day.

Fcg : Fcγ