Cristian Suarez

Profilin Regulates F-Actin Network Homeostasis by Favoring Formin over Arp2/3 Complex

Fission yeast cells use Arp2/3 complex and formin to assemble diverse filamentous actin (F-actin) networks within a common cytoplasm for endocytosis, division, and polarization. Although these homeostatic F-actin networks are usually investigated separately, competition for a limited pool of actin monomers (G-actin) helps to regulate their size and density. However, the mechanism by which G-actin is correctly distributed between rival F-actin networks is not clear. Using a combination of cell biological approaches and in vitro reconstitution of competition between actin assembly factors, we found that the small G-actin binding protein profilin directly inhibits Arp2/3 complex-mediated actin assembly. Profilin is therefore required for formin to compete effectively with excess Arp2/3 complex for limited G-actin and to assemble F-actin for contractile ring formation in dividing cells.

Homeostatic Actin Cytoskeleton Networks Are Regulated by Assembly Factor Competition for Monomers

Controlling the quantity and size of organelles through competition for a limited supply of components is quickly emerging as an important cellular regulatory mechanism. Cells assemble diverse actin filament (F-actin) networks for fundamental processes including division, motility, and polarization. F-actin polymerization is tightly regulated by activation of assembly factors such as the Arp2/3 complex and formins at specific times and places. We directly tested an additional hypothesis that diverse F-actin networks are in homeostasis, whereby competition for actin monomers (G-actin) is critical for regulating F-actin network size. Here we show that inhibition of Arp2/3 complex in the fission yeast Schizosaccharomyces pombe not only depletes Arp2/3-complex-mediated endocytic actin patches, but also induces a dramatic excess of formin-assembled F-actin. Conversely, disruption of formin increases the density of Arp2/3-complex-mediated patches. Furthermore, modification of actin levels significantly perturbs the fission yeast actin cytoskeleton. Increasing actin favors Arp2/3-complex-mediated actin assembly, whereas decreasing actin favors formin-mediated contractile rings. Therefore, the specific actin concentration in a cell is critical, and competition for G-actin helps regulate the proper amount of F-actin assembly for diverse processes.

Rickettsia Sca2 has evolved formin-like activity through a different molecular mechanism

Significance Rickettsia Sca2 mimics eukaryotic formins by promoting actin filament nucleation and elongation to assemble actin comet tails for Rickettsia motility. We show that unlike formins, Sca2 is monomeric, but has N- and C-terminal repeat domains (NRD and CRD) that interact with each other. The structure of NRD reveals a new crescent-like fold. CRD is predicted to share this fold, and might form together with NRD a doughnut-shaped formin-like structure for processive elongation. Between NRD and CRD, proline-rich sequences incorporate profilin-actin for elongation, and WASP-homology 2 (WH2) domains recruit actin monomers for nucleation. Rickettsia has therefore “rediscovered” formin-like actin nucleation and elongation. Sca2 (surface cell antigen 2) is the only bacterial protein known to promote both actin filament nucleation and profilin-dependent elongation, mimicking eukaryotic formins to assemble actin comet tails for Rickettsia motility. We show that Sca2’s functional mimicry of formins is achieved through a unique mechanism. Unlike formins, Sca2 is monomeric, but has N- and C-terminal repeat domains (NRD and CRD) that interact with each other for processive barbed-end elongation. The crystal structure of NRD reveals a previously undescribed fold, consisting of helix–loop–helix repeats arranged into an overall crescent shape. CRD is predicted to share this fold and might form together with NRD, a doughnut-shaped formin-like structure. In between NRD and CRD, proline-rich sequences mediate the incorporation of profilin-actin for elongation, and WASP-homology 2 (WH2) domains recruit actin monomers for nucleation. Sca2’s α-helical fold is unusual among Gram-negative autotransporters, which overwhelmingly fold as β-solenoids. Rickettsia has therefore “rediscovered” formin-like actin nucleation and elongation.

Internetwork competition for monomers governs actin cytoskeleton organization

Cells precisely control the formation of dynamic actin cytoskeleton networks to coordinate fundamental processes, including motility, division, endocytosis and polarization. To support these functions, actin filament networks must be assembled, maintained and disassembled at the correct time and place, and with proper filament organization and dynamics. Regulation of the extent of filament network assembly and of filament network organization has been largely attributed to the coordinated activation of actin assembly factors through signalling cascades. Here, we discuss an intriguing model in which actin monomer availability is limiting and competition between homeostatic actin cytoskeletal networks for actin monomers is an additional crucial regulatory mechanism that influences the density and size of different actin networks, thereby contributing to the organization of the cellular actin cytoskeleton.

ACD toxin-produced actin oligomers poison formin-controlled actin polymerization

A little toxin can do a lot The actin cross-linking domain (ACD) is an actin-specific toxin produced by several bacterial pathogens. Heisler et al. discovered that ACDs pathogenic mechanism involves a highly unusual toxicity amplification cascade. Rather than directly inactivating the actin cytoskeleton, ACD blocks the activity of formins, actin regulatory proteins that play crucial roles in numerous cellular activities. ACD is exceptionally potent, even though its substrate is the most abundant protein of a eukaryotic cell: actin. Science, this issue p. 535 An actin-specific toxin employs actin oligomers to subvert cellular functions at very low doses. The actin cross-linking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin cross-linking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here, we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently “poisoned” the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by using actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses.

A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface

Background: Plasmodium falciparum actin depolymerizing factor 1 (PfADF1) severs actin polymers without stable filament-binding, challenging current models for severing. Results: Cross-linking mass spectrometry of PfADF1 with filamentous actin reveals a novel binding interface required for severing. Conclusion: Filament severing by PfADF1 is via a previously unidentified binding interface. Significance: We propose an alternative mechanism for actin filament severing potentially used across eukaryotic cells. Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.