Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

Actin Dynamics, Architecture, and Mechanics in Cell Motility

Clathrin-mediated endocytosis is a key process in vesicular trafficking that transports a wide range of cargo molecules from the cell surface to the interior. Clathrin-mediated endocytosis was first described over 5 decades ago. Since its discovery, over 50 proteins have been shown to be part of the molecular machinery that generates the clathrin-coated endocytic vesicles. These proteins and the different steps of the endocytic process that they mediate have been studied in detail. However, we still lack a good understanding of how all these different components work together in a highly coordinated manner to drive vesicle formation. Nevertheless, studies in recent years have provided several important insights into how endocytic vesicles are built, starting from initiation, cargo loading and the mechanisms governing membrane bending to membrane scission and the release of the vesicle into the cytoplasm.

Mechanisms of clathrin-mediated endocytosis.

Successful cell division requires the precise and timely coordination of chromosomal, cytoskeletal and membrane trafficking events. These processes are regulated by the competing actions of protein kinases and phosphatases. Aurora B is one of the most intensively studied kinases. In conjunction with inner centromere protein (INCENP), borealin (also known as Dasra) and survivin it forms the chromosomal passenger complex (CPC). This complex targets to different locations at differing times during mitosis, where it regulates key mitotic events: correction of chromosome-microtubule attachment errors; activation of the spindle assembly checkpoint; and construction and regulation of the contractile apparatus that drives cytokinesis. Our growing understanding of the CPC has seen it develop from a mere passenger riding on the chromosomes to one of the main controllers of mitosis.

/pdf/the-chromosomal-passenger-complex-cpc-from-easy-rider-to-the-1sga3ojqcp.pdf

The Chromosomal Passenger Complex (CPC): From Easy Rider to the Godfather of Mitosis

Inorganic polyphosphate (Poly P) is a polymer of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds as in ATP. Found in abundance in all cells in nature, it is unique in its likely role in the origin and survival of species. Here, we present extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth. Beyond that, Poly P has proved in a variety of ways to be essential for growth of cells, their responses to stresses and stringencies, and the virulence of pathogens. In this review, we pay particular attention to the enzyme, polyphosphate kinase 1 (Poly P kinase 1 or PPK1), responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens. Mutants lacking PPK1 are defective in motility, quorum sensing, biofilm formation, and virulence. Structural studies are cited that reveal the conserved ATP-binding site of PPK1 at atomic resolution and reveal that the site can be blocked with minute concentrations of designed inhibitors. Another widely conserved enzyme is PPK2, which has distinctive kinetic properties and is also implicated in the virulence of some pathogens. Thus, these enzymes, absent in yeast and animals, are novel attractive targets for treatment of many microbial diseases. Still another enzyme featured in this review is one discovered in Dictyostelium discoideum that becomes an actin-like fiber concurrent with the synthesis, step by step, of a Poly P chain made from ATP. The Poly P-actin fiber complex, localized in the cell, lengthens and recedes in response to metabolic signals. Homologs of DdPPK2 are found in pathogenic protozoa and in the alga Chlamydomonas. Beyond the immediate relevance of Poly P as a target for anti-infective drugs, a large variety of cellular operations that rely on Poly P will be considered.

Inorganic polyphosphate: essential for growth and survival.

Organisms from all domains of life depend on filaments of the protein actin to provide structure and to support internal movements. Many eukaryotic cells use forces produced by actin polymerization for their motility, and myosin motor proteins use ATP hydrolysis to produce force on actin filaments. Actin polymerizes spontaneously, followed by hydrolysis of a bound adenosine triphosphate (ATP). Dissociation of the γ-phosphate prepares the polymer for disassembly. This review provides an overview of the properties of actin and shows how dozens of proteins control both the assembly and disassembly of actin filaments. These players catalyze nucleotide exchange on actin monomers, initiate polymerization, promote phosphate dissociation, cap the ends of polymers, cross-link filaments to each other and other cellular components, and sever filaments.

/pdf/actin-and-actin-binding-proteins-bqre8kx297.pdf

Actin and Actin-Binding Proteins

When tagged with a fluorescent protein, actin is not fully functional, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP-LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP-LifeAct and LifeAct-mCherry bind actin filaments with Kd values of ∼10 μM. LifeAct-mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct-mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts.

/pdf/avoiding-artefacts-when-counting-polymerized-actin-in-live-pgy183b8vv.pdf

Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins

We created two new mutants of fission yeast cofilin to investigate why cytokinesis in many organisms depends on this small actin-binding protein. These mutant cofilins bound actin monomers normally, but bound and severed ADP-actin filaments much slower than wild-type cofilin. Cells depending on mutant cofilins condensed nodes, precursors of the contractile ring, into clumps rather than rings. Starting from clumped nodes, mutant cells slowly assembled rings from diverse intermediate structures including spiral strands containing actin filaments and other contractile ring proteins. This process in mutant cells depended on α-actinin. These slowly assembled contractile rings constricted at a normal rate but with more variability, indicating ring constriction is not very sensitive to defects in severing by cofilin. Computer simulations of the search-capture-pull and release model of contractile ring formation predicted that nodes clump when the release step is slow, so cofilin severing of actin filament connections between nodes likely contributes to the release step.

/pdf/actin-filament-severing-by-cofilin-is-more-important-for-2748f5o3i8.pdf

Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring

Formins filter modified actin subunits during processive elongation.

https://pollardlab.yale.edu/sites/default/files/files/news/Chen7813.pdf

Qian Chen

Papers

Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins

Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring

Formins filter modified actin subunits during processive elongation.

Actin filament severing by cofilin dismantles actin patches and produces mother filaments for new patches.

Aip1 Promotes Actin Filament Severing by Cofilin and Regulates Constriction of the Cytokinetic Contractile Ring