Physical Constraints and Forces Involved in Phagocytosis

Abstract

Phagocytosis is a specialized process that enables cellular ingestion and clearance of microbes, dead cells and tissue debris that are too large for other endocytic routes. As such, it is an essential component of tissue homeostasis and the innate immune response, and also provides a link to the adaptive immune response. However, ingestion of large particulate materials represents a monumental task for phagocytic cells. It requires profound reorganization of the cell morphology around the target in a controlled manner, which is limited by biophysical constraints. Experimental and theoretical studies have identified critical aspects associated with the interconnected biophysical properties of the receptors, the membrane, and the actin cytoskeleton that can determine the success of large particle internalization. In this review, we will discuss the major physical constraints involved in the formation of a phagosome. Focusing on two of the most-studied types of phagocytic receptors, the Fcγ receptors and the complement receptor 3 (αMβ2 integrin), we will describe the complex molecular mechanisms employed by phagocytes to overcome these physical constraints.

Keywords: Fc receptors; actin dynamics; cell mechanics; integrins; membrane; phagocytosis; receptor diffusion.

Figure 1

Actin-based internalization mechanisms of large particulate materials. The trigger mechanism (Left) enables internalization in an adhesion-independent manner. Macropinocytosis is a trigger mechanism typically induced by growth factors, such as MCSF or EGF. Bacterial pathogens like Shigella and Salmonella induce their internalization via a trigger mechanism using a type III secretion system to inject effectors inside the host cell, which induce actin polymerization to induce local ruffle formation which surround and engulf the bacteria. Numerous viruses also enter their host cell through macropinocytosis. The zipper mechanism (Right) requires adhesion to host cell receptors along the entire surface of the particle. Converging evidence demonstrates that phagocytosis occurs through a zipper mechanism.

Figure 2

Sequence of events involved in particle uptake by phagocytosis. (1) Phagocytic receptors (dark blue) diffuse along the plane of the plasma membrane and bind ligands (yellow) at the surface of the particulate target (purple). (2) Ligand binding and clustering of the receptors elicits signaling that activates actin polymerization, which generate a protrusive force against the plasma membrane. (3) Anchorage of the actin cytoskeleton to ligand-bound receptors on the particle surface by coupling or tethering proteins, tangentially to the direction of actin polymerization, enables membrane protrusions to extend over the particle surface. (4) For large particulate targets, mobilization of membrane reservoirs from surface folds of the plasma membrane and intracellular vesicles provides the required membrane surface area to envelop the particle. (5) Once the particle is fully enveloped and the protrusions reach a meeting point, membrane fission enables the separation of the phagosome from the plasma membrane.

Figure 3

Schematic of the structures of the integrin αMβ2, the Fcγ Receptor IIA and the phosphatase CD45. The integrin αMβ2 (left) exists in at least three distinct conformations: a bent-closed conformation associated with a low affinity for its ligands, an extended-closed conformation associated with an intermediate affinity, and an extended-open conformation associated with a high affinity for its ligands. The current model suggests that integrins are maintained in an autoinhibited bent conformation by the interaction of the cytosolic domains of the α and the β chains. The switch from a bent to an extended conformation requires binding of Talin to the cytosolic domain of the β chain. The pulling force generated by the actin cytoskeleton through Talin induces the open conformation, when the integrin is attached to an immobile ligand. The ligand iC3b binds through its TED domain to the αI domain of αM. Some evidence suggest that the C345C domain could associate with the βI domain of β2, in addition to the TED-αI association. Contrary to integrins, the structure of FcγRIIA shows no conformational change upon binding to an immunoglobulin G. Average heights estimated from the membrane surface for these receptors in each conformation and for the RO isoform of CD45 are shown. For the extended conformations of αMβ2, the indicated height corresponds to the height of the β-propeller. Talin was not schematized to scale, but its estimated length at a focal adhesion is indicated.

Figure 4

Proposed model of the cellular forces involved during internalization by phagocytosis. Phagosome formation is driven by a protrusive force (red arrows), generated by the polymerization of actin filaments, directed along the particle by an attractive force (purple arrows), and coupling proteins that anchor the actin cytoskeleton to the cell-particle interface. The protrusive force works against the surface tension, composed of the cortical tension (pink arrows) and the membrane tension (blue arrows). The rate of deformation of the cell is determined by the ratio of the surface tension and the cytoplasm viscosity, while the surface tension effectively propels the particle inward. The in-line tension of the plasma membrane is compensated by the flattening of surface folds of the membrane and the exocytosis of intracellular vesicles.

Figure 5

Models of the actin cytoskeleton organization at the front of a migrating cell and at the phagocytic cup. The formation of a branched network of actin filaments (red) is mediated by the Arp2/3 complex (green ellipse), which is activated at the plasma membrane by an NPF recruited by an active Rho GTPase (pink double-circle). Actin dynamics are regulated by capping proteins (orange ellipse), elongation factors, debranching, severing, and monomer binding proteins (not represented for clarity). Formation of linear actin structures, such as the stress fibers and transverse arcs at the lamella involve the bundling proteins α-Actinin (light blue rod) and Fascin (dark blue rod), actin nucleation by formins (red circle) and Myosin II mini-filaments (black dumbbell). These actin structures are stabilized on the substratum by adhesions (purple). At the phagocytic cup, adhesions are mediated by Fcγ receptors, β2 integrins or other phagocytic receptors.

Figure 6

The molecular clutch model in αMβ2-mediated phagocytosis. The Arp2/3 complex (light green) nucleates actin (red) polymerization at the leading edge of the phagocytic cup. The addition of new monomers to the filaments at the membrane generates forces that push against the plasma membrane, leading to an equilibrium between membrane protrusion and the retrograde flow of actin filaments. Coupling of iC3b (yellow) -bound αMβ2 integrins (purple and pink) to actin filaments by Talin (dark green), transmits mechanical tension that switches the integrins into an extended-open conformation and provides traction on the particle. Stretching of Talin reveal Vinculin (blue) binding sites and the phosphorylation of Paxillin (orange) by tyrosine kinases (purples). This leads to the recruitment of Vinculin, which reinforces the molecular clutch to prevent its slippage, reducing the retrograde flow of actin and increasing traction and forward protrusion. In addition, contractility mediated by Myosin II (black) could increase tension across Talin and increase the actin retrograde flow and traction onto the particle, while reducing the protrusion of the phagocytic cup edge.