Gelsolin

[Shu Manwill]

Gelsolinis an actin-binding protein that is a key regulator of actin filament assembly and disassembly. Gelsolin is one of the most potent members of the actin-severing gelsolin/villin superfamily, as it severs with nearly 100% efficiency.[4][5]

Cellular gelsolin, found within the cytosol and mitochondria,[6] has a closely related secreted form, Plasma gelsolin, that contains an additional 24 AA N-terminal extension.[7][8] Plasma gelsolin's ability to sever actin filaments helps the body recover from disease and injury that leaks cellular actin into the blood. Additionally it plays important roles in host innate immunity, activating macrophages and localizing of inflammation.

Gelsolin is an 82-kD protein with six homologous subdomains, referred to as S1-S6. Each subdomain is composed of a five-stranded β-sheet, flanked by two α-helices, one positioned perpendicular with respect to the strands and one positioned parallel. The β-sheets of the three N-terminal subdomains (S1-S3) join to form an extended β-sheet, as do the β-sheets of the C-terminal subdomains (S4-S6).[9]

Among the lipid-binding actin regulatory proteins, gelsolin (like cofilin) preferentially binds polyphosphoinositide (PPI).[10] The binding sequences in gelsolin closely resemble the motifs in the other PPI-binding proteins.[10]

Gelsolin's activity is stimulated by calcium ions (Ca2+).[5] Although the protein retains its overall structural integrity in both activated and deactivated states, the S6 helical tail moves like a latch depending on the concentration of calcium ions.[11] The C-terminal end detects the calcium concentration within the cell. When there is no Ca2+ present, the tail of S6 shields the actin-binding sites on one of S2's helices.[9] When a calcium ion attaches to the S6 tail, however, it straightens, exposing the S2 actin-binding sites.[11] The N-terminal is directly involved in the severing of actin. S2 and S3 bind to the actin before the binding of S1 severs actin-actin bonds and caps the barbed end.[10]

Gelsolin also inhibits apoptosis by stabilizing the mitochondria.[6] Prior to cell death, mitochondria normally lose membrane potential and become more permeable. Gelsolin can impede the release of cytochrome C, obstructing the signal amplification that would have led to apoptosis.[13]

Sequence comparisons indicate an evolutionary relationship between gelsolin, villin, fragmin, and severin.[17] Six large repeating segments occur in gelsolin and villin, and 3 similar segments in severin and fragmin. The multiple repeats are related in structure (but barely in sequence) to the ADF-H domain, forming a superfamily (InterPro: IPR029006). The family appears to have evolved from an ancestral sequence of 120 to 130 amino acid residues.[17][4]

Gelsolin is a cytoplasmic, calcium-regulated, actin-modulating protein that binds to the barbed ends of actin filaments, preventing monomer exchange (end-blocking or capping).[19] It can promote nucleation (the assembly of monomers into filaments), as well as sever existing filaments. In addition, this protein binds with high affinity to fibronectin. Plasma gelsolin and cytoplasmic gelsolin are derived from a single gene by alternate initiation sites and differential splicing.[7]

Recombinant antibodies offer several key advantages compared to traditional antibodies. These include superior lot-to-lot consistency, continuous supply, and animal-free manufacturing. As such, recombinant antibodies are seeing increased use for scientific research, especially as a means of addressing the ongoing reproducibility crisis.

Traditional polyclonal and monoclonal antibodies are the product of normal B cell development and genetic recombination. They are generated by immunizing an animal with an antigen to elicit an immune response. While polyclonal antibodies are secreted by many different B cell clones and recognize multiple antigenic epitopes, monoclonals originate from a single B cell clone and are specific for just one epitope.

Recombinant antibodies are monoclonal, but their production involves in vitro genetic manipulation. After cloning the antibody genes into an expression vector, this is then transfected into an appropriate host cell line for antibody expression. Mammalian cell lines are most commonly used for recombinant antibody production, although cell lines of bacterial, yeast, or insect origin are also suitable.

Because recombinant antibody production involves sequencing the antibody light and heavy chains, it is a highly controlled and reliable process. In contrast, hybridoma-based systems for producing monoclonal antibodies are subject to genetic drift and instability, increasing the potential for lot-to-lot variability or loss of antibody expression. Recombinant antibodies are highly consistent from lot to lot, thereby ensuring reproducible experimental results.

In vitro methods for producing antibodies are amenable to large-scale production, meaning antibody availability is unlikely to become a limiting factor. Moreover, since the recombinant antibody sequence is known, continuity of supply is assured; in situations where an antibody will be used to support large, long-term studies, this can be an especially critical factor.

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95-98C). Cool slides on bench top for 30 min.

NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.

Gelsolin (actin-depolymerizing factor, ADF, AGEL, Brevin) is an 83 kDa protein that shares structural and functional homology to villin and adseverin/scinderin (1,2). Gelsolin plays an important role in actin filament assembly by capping and severing actin proteins in a Ca2+-dependent manner (3,4). Gelsolin is important for cellular events (e.g., membrane ruffling, chemotaxis, ciliogenesis) that require cytoskeletal remodeling (3). Accordingly, cells from gelsolin knockout mice exhibit motility defects, including a failure to ruffle in response to growth factor stimulation (5,6). In humans, defects in gelsolin have been linked to amyloidosis type 5 (AMYL5), a hereditary disease characterized by cranial neuropathy, which appears to result from gelsolin amyloid deposition (7).

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Biomechanical stress and cytoskeletal remodeling are key determinants of cellular homeostasis and tissue responses to mechanical stimuli and injury. Here we document the increased activity of gelsolin, an actin filament severing and capping protein, in failing human hearts. Deletion of gelsolin prevents biomechanical stress-induced adverse cytoskeletal remodeling and heart failure in mice. We show that phosphatidylinositol (3,4,5)-triphosphate (PIP3) lipid suppresses gelsolin actin-severing and capping activities. Accordingly, loss of PI3Kα, the key PIP3-producing enzyme in the heart, increases gelsolin-mediated actin-severing activities in the myocardium in vivo, resulting in dilated cardiomyopathy in response to pressure-overload. Mechanical stretching of adult PI3Kα-deficient cardiomyocytes disrupts the actin cytoskeleton, which is prevented by reconstituting cells with PIP3. The actin severing and capping activities of recombinant gelsolin are effectively suppressed by PIP3. Our data identify the role of gelsolin-driven cytoskeletal remodeling in heart failure in which PI3Kα/PIP3 act as negative regulators of gelsolin activity.

Heart failure (HF) is driven by a complex series of signaling and injury pathways that lead to maladaptive cardiac remodeling1,2. Hypertension, which leads to increased afterload and biomechanical stress on the heart, is the most important cause of HF2,3. Biomechanical stress is converted to intracellular signals through mechanotransduction processes4,5,6; remodeling of the cytoskeleton is a central feature of these processes. However, the regulation of these processes and their contribution to HF is poorly understood. Gelsolin is a Ca2+-regulated actin filament severing and capping protein, that is widely expressed in a variety of tissues including the heart, brain, immune cells, and various cancer tissues7. Importantly, gelsolin favors actin depolymerization by virtue of both its actin-severing activity and its ability to cap the barbed ends of actin filaments, resulting in reduced actin polymerization. Gelsolin has a high-positive charge and contains multiple binding sites for Ca2+ and phosphatidylinositol lipids7,8.

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