Actin in vitro reconstitution

Actin, the most abundant protein in cells, polymerizes into filaments that play key roles in many cellular dynamics. To understand cell dynamics and functions, it is essential to examine the cytoskeleton structure organized by actin and actin-binding proteins. Here, we pave the way for determining the molecular assembly of the actin cytoskeleton using direct photonic in situ analysis, providing the photoluminescence characteristics of actin as a function of filament length and bundling, without labeling. In experiments for monomeric and filamentous actin reconstituted in vitro, structural forms of actin are identified from the peak positions and intensities of photoluminescence...

Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis...

The complex architecture of neuronal networks in the brain requires tight control of the actin cytoskeleton. The actin nucleator Cobl is critical for neuronal morphogenesis. Here we reveal that Cobl is controlled by arginine methylation. Coprecipitations, coimmunoprecipitations, cellular reconstitutions, and in vitro reconstitutions demonstrated that Cobl associates with the protein arginine methyltransferase PRMT2 in a Src Homology 3 (SH3) domain-dependent manner and that this promotes methylation of Cobl's actin nucleating C-terminal domain...

Chronic lung diseases (CLDs) are regulated by complex interactions between many different cell types residing in lung airway tissues. Specifically, interactions between airway epithelial cells (ECs) and airway smooth muscle cells (SMCs) have been shown in part to play major roles in the pathogenesis of CLDs, but the underlying molecular mechanisms are not well understood. To advance our understanding of lung pathophysiology and accelerate drug development processes, new innovative in vitro tissue models are needed that can reconstitute the complex in vivo microenvironment of human lung tissues...

In living matter, shape fluctuations induced by acto-myosin are usually studied in vitro via reconstituted gels, whose properties are controlled by changing the concentrations of actin, myosin, and cross-linkers. Such an approach deliberately avoids consideration of the complexity of biochemical signaling inherent to living systems. Acto-myosin activity inside living cells is mainly regulated by the Rho signaling pathway, which is composed of multiple layers of coupled activators and inhibitors. Here, we investigate how such a pathway controls the dynamics of confluent epithelial tissues by tracking the displacements of the junction points between cells...

Myosin-driven contraction of the actin cytoskeleton is at the base of cell and tissue morphogenesis. At the molecular level, myosin motors drive contraction by sliding actin filaments past one another using energy produced by ATP hydrolysis. How this microscopic sliding activity gives rise to cell-scale contractions has been an active research question first in muscle cells, and over the last few decades in non-muscle cells. While many early investigations focused on myosin motor activity, increasingly, the nanoscale architecture of the actin network emerges as a key regulator of contractility...

Although cytoskeletal networks are interpenetrating and interacting in living cells, very little is understood as to the effect their interaction has on their properties. Here, as a step towards elucidating the synergistic cellular role of these structural proteins, we investigate isolated keratin and actin composites and show how the in vitro network formation of keratin influences the properties of actin networks and vice versa. By encapsulating purified composite networks into vesicles and separating the time scales of network formation we are able to demonstrate that the actin network stabilizes keratin networks by providing an elastic resistance to their collapse in vitro...

The interaction between actin filaments and myosin molecular motors is a power source of a variety of cellular functions including cell division, cell motility, and muscular contraction. In vitro motility assay examines actin filaments interacting with myosin molecules that are adhered to a substrate (e.g., glass surface). This assay has been the standard method of studying the molecular mechanisms of contraction under an optical microscope. While the force generation has been measured through an optically trapped bead to which an actin filament is attached, a force vector vertical to the glass surface has been largely ignored with the in vitro motility assay...

T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells...

Formins polymerize actin filaments for the cytokinetic contractile ring. Using in vitro reconstitution of fission yeast contractile ring precursor nodes containing formins and myosin, a new study shows that formin-mediated polymerization is strongly inhibited upon the capture and pulling of actin filaments by myosin, a result that has broad implications for cellular mechanosensing.

Cytokinesis physically separates dividing cells by forming a contractile actomyosin ring. The fission yeast contractile ring has been proposed to assemble by Search-Capture-Pull-Release from cytokinesis precursor nodes that include the molecular motor type-II myosin Myo2 and the actin assembly factor formin Cdc12. By successfully reconstituting Search-Capture-Pull in vitro, we discovered that formin Cdc12 is a mechanosensor, whereby myosin pulling on formin-bound actin filaments inhibits Cdc12-mediated actin assembly...

The growth of branched actin networks powers cell-edge protrusions and motility. A heterogeneous density of actin, which yields to a tunable cellular response, characterizes these dynamic structures. We study how actin organization controls both the rate and the steering during lamellipodium growth. We use a high-resolution surface structuration assay combined with mathematical modeling to describe the growth of a reconstituted lamellipodium. We demonstrate that local monomer depletion at the site of assembly negatively impacts the network growth rate...

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5...

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane...

Clathrin/adaptor protein-1-coated carriers connect the secretory and the endocytic pathways. Carrier biogenesis relies on distinct protein networks changing membrane shape at the trans-Golgi network, each regulating coat assembly, F-actin-based mechanical forces, or the biophysical properties of lipid bilayers. How these different hubs are spatiotemporally coordinated remains largely unknown. Using in vitro reconstitution systems, quantitative proteomics, and lipidomics, as well as in vivo cell-based assays, we characterize the protein networks controlling membrane lipid composition, membrane shape, and carrier scission...

BACKGROUND: Cellular dynamics depend on cytoskeletal filaments and motor proteins. Collective movements of filaments driven by motor proteins are observed in the presence of dense filaments in in vitro systems. As multiple macromolecules exist within cells and the physiological ionic conditions affect their interactions, crowding might contribute to ordered cytoskeletal architecture because of collective behavior. METHODS: Using an in vitro reconstituted system, we observed the emergence of stripe patterns resulting from collective actin filament streaming driven by myosin motors in the presence of the crowding agent, methylcellulose (MC)...

The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments...

Biochemical reconstitution has served as an important tool for understanding the mechanisms of many cellular processes including DNA replication, transcription, translation, vesicle trafficking, and ubiquitin-mediated proteolysis. Here, we demonstrate that biochemical reconstitution can be applied to studying a complex signaling pathway involving as many as 12 proteins or protein complexes acting at the surface of model membranes. We show that a temporal sequence of events in activated T cells beginning with phosphorylation of the T cell receptor and culminating in the activation of actin polymerization can be replicated in vitro...

Hypertrophic cardiomyopathy (HCM) is a severe heart disease caused by missense mutations in genes encoding sarcomeric proteins of cardiac muscle. Many of these mutations are identified in the gene encoding the cardiac isoform of tropomyosin (Tpm), an α-helical coiled-coil actin-binding protein that plays a key role in Ca(2+)-regulated contraction of cardiac muscle. We employed various methods to characterize structural and functional features of recombinant human Tpm species carrying HCM mutations that lie either within the troponin T-binding region in the C-terminal part of Tpm (E180G, E180V, and L185R) or near this region (I172T)...

The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers...