Principles of Fibrinogen Fiber Assembly In Vitro

SummaryMultimerin 1 (MMRN1) is a large, soluble, polymeric, factor V binding protein and member of the EMILIN protein family.In vivo, MMRN1 is found in platelets, megakaryocytes, endothelium and extracellular matrix fibers, but not in plasma. To address the mechanism of MMRN1 binding to activated platelets and endothelial cells, we investigated the identity of the major MMRN1 receptors on these cells using wild-type and RGE-forms of recombinant MMRN1. Ligand capture, cell adhesion, ELISA and flow cytometry analyses of platelet-MMRN1 binding, indicated that MMRN1 binds to integrins αIIbβ3 and αvβ3. Endothelial cell binding to MMRN1 was predominantly mediated by αvβ3 and did not require the MMRN1 RGD site or cellular activation. Like many other αvβ3 ligands, MMRN1 had the ability to support adhesion of additional cell types, including stimulated neutrophils. Expression studies, using a cell line capable of endothelial-like MMRN1 processing, indicated that MMRN1 adhesion to cellular receptors enhanced its extracellular matrix fiber assembly. These studies implicate integrin-mediated binding in MMRN1 attachment to cells and indicate that MMRN1 is a ligand for αIIbβ3 and αvβ3.

Download Full-text

67-kD elastin-binding protein is a protective "companion" of extracellular insoluble elastin and intracellular tropoelastin.

The Journal of Cell Biology ◽

10.1083/jcb.126.2.563 ◽

1994 ◽

Vol 126 (2) ◽

pp. 563-574 ◽

Cited By ~ 121

Author(s):

A Hinek ◽

M Rabinovitch

Keyword(s):

Binding Protein ◽

Light And Electron Microscopy ◽

Serine Proteinase ◽

Elastic Fibers ◽

Fiber Assembly ◽

Elastin Fiber ◽

Alternatively Spliced ◽

Elastin Binding Protein ◽

Highly Hydrophobic

The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S-GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S-GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.

Download Full-text

Engineered Whole Cut Meats Assembled of Cell Fibers Constructed by Tendon-Gel Integrated Bioprinting

10.21203/rs.3.rs-91978/v1 ◽

2020 ◽

Author(s):

Dong-Hee Kang ◽

Fiona Louis ◽

Hao Liu ◽

Hiroshi Shimoda ◽

Yasutaka Nishiyama ◽

...

Keyword(s):

Blood Capillary ◽

High Demand ◽

Fiber Assembly ◽

Cultured Meat ◽

Bovine Cell ◽

Beef Steak ◽

Engineered Structures ◽

Histological Images ◽

Contraction And Relaxation

Abstract With the current interest in artificial meat, mammalian cell-based cultured meat has mostly been in minced form. There is thus still a high demand for artificial steak-like meat. Herein, we demonstrate in vitro construction of engineered steak-like meat assembled of three types of edible bovine cell fibers, such as skeletal muscle, adipose, and blood capillary fabricated by tendon-gel integrated printing (TIP) technology. Because actual meat is an anisotropically aligned assembly of the fibers connected to tendon for the actions of contraction and relaxation, TIP was discovered to construct the fiber assembly connecting tendon gels with engineered structures. In this study, a total of 72 fibers comprising 42 muscle, 28 adipose, and 2 blood capillary were constructed by TIP and subsequently assembled to fabricate a steak-like meat with a diameter of 5 mm and a length of 10 mm by consulting histological images of actual Wagyu beef steak. The TIP discovered here could be a powerful manufacturing technology for fabrication of the desired types of steak-like cultured meats.

Download Full-text

Citron Kinase, a Rho-dependent Kinase, Induces Di-phosphorylation of Regulatory Light Chain of Myosin II

Molecular Biology of the Cell ◽

10.1091/mbc.e02-07-0427 ◽

2003 ◽

Vol 14 (5) ◽

pp. 1745-1756 ◽

Cited By ~ 143

Author(s):

Shigeko Yamashiro ◽

Go Totsukawa ◽

Yoshihiko Yamakita ◽

Yasuharu Sasaki ◽

Pascal Madaule ◽

...

Keyword(s):

Light Chain ◽

Myosin Ii ◽

Specific Inhibitor ◽

Kinase Domain ◽

Regulatory Light Chain ◽

Myosin Phosphatase ◽

Fiber Assembly ◽

Citron Kinase

Citron kinase is a Rho-effector protein kinase that is related to Rho-associated kinases of ROCK/ROK/Rho-kinase family. Both ROCK and citron kinase are suggested to play a role in cytokinesis. However, no substrates are known for citron kinase. We found that citron kinase phosphorylated regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase did not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. We found that the expression of the kinase domain of citron kinase resulted in an increase in MLC di-phosphorylation. Furthermore, the kinase domain was able to increase di-phosphorylation and restore stress fiber assembly even when ROCK was inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increased di-phosphorylation during cytokinesis. These observations suggest that citron kinase phosphorylates MLC to generate di-phosphorylated MLC in vivo. Although both mono- and di-phosphorylated MLC were found in cleavage furrows, di-phosphorylated MLC showed more constrained localization than did mono-phosphorylated MLC. Because citron kinase is localized in cleavage furrows, citron kinase may be involved in regulating di-phosphorylation of MLC during cytokinesis.

Download Full-text

Hydrostatic Pressure Shows That Lamellipodial Motility in Ascaris Sperm Requires Membrane-associated Major Sperm Protein Filament Nucleation and Elongation

The Journal of Cell Biology ◽

10.1083/jcb.140.2.367 ◽

1998 ◽

Vol 140 (2) ◽

pp. 367-375 ◽

Cited By ~ 24

Author(s):

Thomas M. Roberts ◽

E.D. Salmon ◽

Murray Stewart

Keyword(s):

Plasma Membrane ◽

Hydrostatic Pressure ◽

Leading Edge ◽

Major Sperm Protein ◽

Sperm Protein ◽

Fiber Assembly ◽

Fiber Growth ◽

Filament Assembly

Sperm from nematodes use a major sperm protein (MSP) cytoskeleton in place of an actin cytoskeleton to drive their ameboid locomotion. Motility is coupled to the assembly of MSP fibers near the leading edge of the pseudopod plasma membrane. This unique motility system has been reconstituted in vitro in cell-free extracts of sperm from Ascaris suum: inside-out vesicles derived from the plasma membrane trigger assembly of meshworks of MSP filaments, called fibers, that push the vesicle forward as they grow (Italiano, J.E., Jr., T.M. Roberts, M. Stewart, and C.A. Fontana. 1996. Cell. 84:105–114). We used changes in hydrostatic pressure within a microscope optical chamber to investigate the mechanism of assembly of the motile apparatus. The effects of pressure on the MSP cytoskeleton in vivo and in vitro were similar: pressures >50 atm slowed and >300 atm stopped fiber growth. We focused on the in vitro system to show that filament assembly occurs in the immediate vicinity of the vesicle. At 300 atm, fibers were stable, but vesicles often detached from the ends of fibers. When the pressure was dropped, normal fiber growth occurred from detached vesicles but the ends of fibers without vesicles did not grow. Below 300 atm, pressure modulates both the number of filaments assembled at the vesicle (proportional to fiber optical density and filament nucleation rate), and their rate of assembly (proportional to the rates of fiber growth and filament elongation). Thus, fiber growth is not simply because of the addition of subunits onto the ends of existing filaments, but rather is regulated by pressure-sensitive factors at or near the vesicle surface. Once a filament is incorporated into a fiber, its rates of addition and loss of subunits are very slow and disassembly occurs by pathways distinct from assembly. The effects of pressure on fiber assembly are sensitive to dilution of the extract but largely independent of MSP concentration, indicating that a cytosolic component other than MSP is required for vesicle-association filament nucleation and elongation. Based on these data we present a model for the mechanism of locomotion-associated MSP polymerization the principles of which may apply generally to the way cells assemble filaments locally to drive protrusion of the leading edge.

Download Full-text

Substratum-induced stress fiber assembly in vascular endothelial cells during spreading in vitro

Journal of Cell Science ◽

10.1242/jcs.95.3.507 ◽

1990 ◽

Vol 95 (3) ◽

pp. 507-520

Author(s):

J.C. Yost ◽

I.M. Herman

Keyword(s):

Type Iv Collagen ◽

Vascular Endothelial Cells ◽

Subcellular Fractionation ◽

Stress Fiber ◽

Cytoskeletal Proteins ◽

Acid Extraction ◽

Fiber Assembly ◽

Type Iv ◽

Collagen Substratum

We tested whether aortic endothelial cell (EC)-synthesized substrata, which modulate smooth muscle cell proliferation and EC motility following injury, could influence EC actin cytoskeleton and spreading in vitro. A partial characterization of the substrata indicates that the substratum prepared by deoxycholic acid extraction (DOC-derived substratum) is enriched with fibronectin and type IV collagen. Substratum prepared by removal of the intact monolayer with 20 mM EGTA in PBS (EGTA-derived substratum) contains fibronectin and heparan sulfate proteoglycan, but no type IV collagen. Morphometric analyses were performed on fixed and cytoskeletal antibody treated EC in order to quantitate the extent of spreading and stress fiber (SF) assembly. Compared to plastic, the DOC-derived substratum, a collagenase-treated DOC-derived substratum (CT-DOC-derived substratum) and the EGTA-derived substratum promote EC spreading 2.3-, 2.9- and 1.7-fold, respectively. In addition, there are 4.2-, 4.1- and 2.0-fold more SF on DOC-, CT-DOC- and EGTA-derived substrata, respectively, when compared to plastic. Subcellular fractionation and immunoprecipitation of cytoskeletal proteins from metabolically labeled EC were performed prior to electrophoresis and fluorography. The DOC-derived substratum increases immunoprecipitable actin and myosin 3- to 4.5-fold in both fractions compared to the EGTA-derived substratum and plastic. Collagenase treatment of the DOC-derived substratum partially inhibits this increase. Cycloheximide treatment prevents the rise in soluble actin and myosin as well as causing a reduction in SF number by 1/2 on the DOC-derived substratum and 2/3 on CT-DOC-derived substratum. We propose that fibronectin-collagen interactions are, in part, responsible for inducing endothelial synthesis of cytoskeletal proteins required for SF assembly. This substratum-induced actin-cytoskeletal reorganization facilitates EC spreading in vitro.

Download Full-text

Characterization of an In Vitro Model of Elastic Fiber Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.10.11.3595 ◽

1999 ◽

Vol 10 (11) ◽

pp. 3595-3605 ◽

Cited By ~ 48

Author(s):

Bruce W. Robb ◽

Hiroshi Wachi ◽

Theresa Schaub ◽

Robert P. Mecham ◽

Elaine C. Davis

Keyword(s):

Elastic Fiber ◽

Electron Microscopic Examination ◽

Northern Analysis ◽

Elastic Fibers ◽

Electron Microscopic ◽

Fiber Assembly ◽

Mammalian Expression ◽

The Matrix ◽

Vitro Model

Elastic fibers consist of two morphologically distinct components: elastin and 10-nm fibrillin-containing microfibrils. During development, the microfibrils form bundles that appear to act as a scaffold for the deposition, orientation, and assembly of tropoelastin monomers into an insoluble elastic fiber. Although microfibrils can assemble independent of elastin, tropoelastin monomers do not assemble without the presence of microfibrils. In the present study, immortalized ciliary body pigmented epithelial (PE) cells were investigated for their potential to serve as a cell culture model for elastic fiber assembly. Northern analysis showed that the PE cells express microfibril proteins but do not express tropoelastin. Immunofluorescence staining and electron microscopy confirmed that the microfibril proteins produced by the PE cells assemble into intact microfibrils. When the PE cells were transfected with a mammalian expression vector containing a bovine tropoelastin cDNA, the cells were found to express and secrete tropoelastin. Immunofluorescence and electron microscopic examination of the transfected PE cells showed the presence of elastic fibers in the matrix. Biochemical analysis of this matrix showed the presence of cross-links that are unique to mature insoluble elastin. Together, these results indicate that the PE cells provide a unique, stable in vitro system in which to study elastic fiber assembly.

Download Full-text

Dissection of the Ascaris Sperm Motility Machinery Identifies Key Proteins Involved in Major Sperm Protein-based Amoeboid Locomotion

Molecular Biology of the Cell ◽

10.1091/mbc.e03-04-0246 ◽

2003 ◽

Vol 14 (12) ◽

pp. 5082-5088 ◽

Cited By ~ 34

Author(s):

Shawnna M. Buttery ◽

Gail C. Ekman ◽

Margaret Seavy ◽

Murray Stewart ◽

Thomas M. Roberts

Keyword(s):

Plasma Membrane ◽

Sperm Motility ◽

Membrane Vesicles ◽

Major Sperm Protein ◽

Plasma Membrane Vesicles ◽

Sperm Protein ◽

Fiber Assembly ◽

A Cell ◽

Fiber Proteins

Although Ascaris sperm motility closely resembles that seen in many other types of crawling cells, the lamellipodial dynamics that drive movement result from modulation of a cytoskeleton based on the major sperm protein (MSP) rather than actin. The dynamics of the Ascaris sperm cytoskeleton can be studied in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibers constructed from bundles of MSP filaments. In addition to ATP, MSP, and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins that orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. Here, we identify a fraction of cytosol that is comprised of a small number of proteins but contains all of the soluble components required to assemble fibers. We have purified two of these proteins, designated MSP fiber proteins (MFPs) 1 and 2 and demonstrated by immunolabeling that both are located in the MSP cytoskeleton in cells and in fibers. These proteins had reciprocal effects on fiber assembly in vitro: MFP1 decreased the rate of fiber growth, whereas MFP2 increased the growth rate.

Download Full-text