Gelsolin Contributes to the Motility of A375 Melanoma Cells and this Activity is Mediated by the Fibrous Extracellular Matrix Protein Profile

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)—an actin-binding protein—levels in melanoma cell lines and GSN’s importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin’s BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN’s influence on parameters associated with cells’ ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin’s BM.

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Engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials

10.1101/2020.08.31.276303 ◽

2020 ◽

Author(s):

Marimikel Charrier ◽

Maria Teresa Orozco-Hidalgo ◽

Nicholas Tjahjono ◽

Dong Li ◽

Sara Molinari ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Caulobacter Crescentus ◽

Extracellular Protein ◽

Extracellular Matrix Protein ◽

High Yield ◽

Type I ◽

Self Healing ◽

Protein Matrix ◽

Living Materials

ABSTRACTThe bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions, but require exogenously added material or have limited programmability. Here we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of elastin-like polypeptide (ELP) hydrogel fused to Supercharged SpyCatcher (SC(-)). This biopolymer was secreted at levels of 60 mg/L, an unprecedented level of biopolymer secretion by a gram-negative bacterium. The ELP domain was swapped with either a crosslinkable variant of ELP or resilin-like polypeptide, demonstrating this system is flexible. The SC(-)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag peptides via its engineered Surface-layer. Our work develops protein design rules for Type I secretion in C. crescentus, and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward towards the formation of cohesive engineered living materials.IMPORTANCEEngineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy intensive processing or have limited tunability. We propose a bacterially-synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step towards understanding the necessary parameters to engineering living cells to autonomously construct ELMs.

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The influence of peptide bioregulators on skin aging in 3D culture model

Russian Journal of Skin and Venereal Diseases ◽

10.18821/1560-9588-2016-19-1-58-63 ◽

2016 ◽

Vol 19 (1) ◽

pp. 58-63 ◽

Cited By ~ 1

Author(s):

K. V Kozhina ◽

E. N Volkova ◽

I. N Saburina ◽

Sergey G. Morozov ◽

I. M Zurina ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

3D Culture ◽

Dermal Fibroblasts ◽

Extracellular Matrix Protein ◽

Cytokeratin 19 ◽

Collagen Type Iv ◽

Cultured Fibroblasts ◽

Study Results ◽

Extracellular Matrix Components

He effect of mesotherapy injection (Meso-Wharton R199TM) on the dermal fibroblasts culture, simulating condition of (mature) aging skin cells are studied. Material and methods. The culture of 4th passage fibroblasts (P4), that corresponds to young skin fibroblasts (control) and the culture of 18th passage fibroblasts (P18), that has all the signs of aging dermal fibroblasts (predominance of large cells, slow cell division) were used. Bioactivity was assessed by cell morphology, epithelium-mesenchyme plasticity and expression of fibroblasts markers: cytokeratin 19, elastin, a-smooth muscle actin (aSMA), PCNA (proliferation marker), collagen types I, III, IV and fibronectin. The formation of spheroids occur when fibroblasts P18 are cultivating with the injection medication, on terms comparable to the formation of spheroids from P4 young fibroblasts. From culture of fibroblasts P18, that was cultured without medication, does not form the full spheroid, but aggregation of cells and their gradual destruction with necrotic masses within the unit are observed. The presence of the medication stimulates the “rejuvenation” of cells and subsequent recovery of the mesenchyme-epithelial plasticity of cultured fibroblasts due to the reduced ability to synthesize sufficient to establish the amount of intercellular contacts the extracellular matrix components (fibronectin and collagen), which affects the ability to form spheroids. Culturing spheroids formed with the medication stimulates expression of elastin, collagen type IV, fibronectin extracellular matrix protein that supports the skin elasticity and superficial cells actively express cytokeratin 19. The study results clearly demonstrate the effectiveness of mesotherapeutic treatment for skin rejuvenation.

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Sphingosine-1-phosphate inhibits extracellular matrix protein-induced haptotactic motility but not adhesion of B16 mouse melanoma cells

FEBS Letters ◽

10.1016/0014-5793(94)80180-0 ◽

1994 ◽

Vol 340 (1-2) ◽

pp. 99-103 ◽

Cited By ~ 17

Author(s):

Yoshito Sadahira ◽

Mingzhe Zheng ◽

Fuqiang Ruan ◽

Sen-itiroh Hakomori ◽

Yasuyuki Igarashi

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Melanoma Cells ◽

Extracellular Matrix Protein ◽

Mouse Melanoma ◽

Sphingosine 1 Phosphate ◽

B16 Mouse Melanoma

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β1 integrin-extracellular matrix protein interaction modulates the migratory response to chemokine stimulation

Biochemistry and Cell Biology ◽

10.1139/o01-026 ◽

2001 ◽

Vol 79 (4) ◽

pp. 399-407 ◽

Cited By ~ 7

Author(s):

Priti S Shenoy ◽

Shashi Uniyal ◽

Kohei Miura ◽

Christopher McColl ◽

Tamas Oravecz ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Collagen Type ◽

Cell Movement ◽

Collagen Type I ◽

Matrix Proteins ◽

Extracellular Matrix Protein ◽

Type I ◽

Migratory Response ◽

Cc Chemokine Receptor 5

It is well established that chemokines have a major role in the stimulation of cell movement on extracellular matrix (ECM) substrates. However, it is also clear that ECM substrates may influence the ability of cells to undergo migration. Using the migration chamber method, we assessed the migratory response of human embryonic kidney-293 (HEK) transfectant cells expressing the CC chemokine receptor 5 (CCR5) (HEK-CCR5) to stimulation by chemokines (macrophage inflamatory protein (MIP)-1α, MIP-1β, and regulated on activation normal-T cell expressed and secreted (RANTES)) on ECM substrates (collagen type I and fibronectin). Using filters coated with collagen (20 µg/mL), results showed that the chemokines differed in their ability to elicit cell movement according to the order MIP-1β > RANTES [Formula: see text] MIP-1α. In contrast, using filters coated with fibronectin (20 µg/mL), all three chemokines were similar in their ability to stimulate migration of HEK-CCR5 cells. In addition, the migratory response with respect to the concentrations of ECM substrates appeared biphasic; thus, chemokine-stimulated cell movement was inhibited at high ECM concentrations (100 µg/mL). To determine the involvement of β1 integrins, results showed that the migratory response to chemokine stimulation on collagen was largely inhibited by monoclonal antibody (mAb) to α2β1; however, complete inhibition required a combination of mAbs to α1β1 and α2β1. In comparison, migration on fibronectin was inhibited by mAb to α3β1 and α5β1. Our results suggest that the migratory response to CCR5 stimulation may vary quantitatively with both the CCR5 ligand (MIP-1α, MIP-1β, and RANTES), as well as the nature and concentration of the ECM substrate involved.Key words: chemokines, integrins, cell movement, extracellular matrix proteins, CCR5.

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The bspA Locus of Lactobacillus fermentum BR11 Encodes an l-Cystine Uptake System

Journal of Bacteriology ◽

10.1128/jb.181.7.2192-2198.1999 ◽

1999 ◽

Vol 181 (7) ◽

pp. 2192-2198 ◽

Cited By ~ 47

Author(s):

Mark S. Turner ◽

Tonia Woodberry ◽

Louise M. Hafner ◽

Philip M. Giffard

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Binding Proteins ◽

Type I Collagen ◽

Lactobacillus Reuteri ◽

Binding Protein ◽

Lactobacillus Fermentum ◽

Extracellular Matrix Protein ◽

Type I ◽

Uptake System

ABSTRACT BspA is a basic surface-exposed protein from Lactobacillus fermentum BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, fromLactobacillus reuteri. Compared with the database ofEscherichia coli proteins, BspA is most similar to thel-cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take upl-cystine. Uptake rates of l-glutamine,l-histidine, and l-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of L. fermentum BR11 with l-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed. L. fermentum BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.

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Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials

mSystems ◽

10.1128/msystems.00903-20 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Maria Teresa Orozco-Hidalgo ◽

Marimikel Charrier ◽

Nicholas Tjahjono ◽

Robert F. Tesoriero ◽

Dong Li ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Caulobacter Crescentus ◽

Design Guidelines ◽

Extracellular Protein ◽

Extracellular Matrix Protein ◽

Type I ◽

Protein Matrix ◽

Content Type ◽

Living Materials

ABSTRACT The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions but require exogenously added material or have limited programmability. Here, we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of an elastin-like polypeptide (ELP) hydrogel fused to supercharged SpyCatcher [SC(−)]. This biopolymer was secreted at levels of 60 mg/liter, an unprecedented level of biomaterial secretion by a native type I secretion apparatus. The ELP domain was swapped with either a cross-linkable variant of ELP or a resilin-like polypeptide, demonstrating this system is flexible. The SC(−)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag (ST) peptides via its engineered surface layer. Our work develops protein design guidelines for type I secretion in C. crescentus and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward toward the formation of cohesive engineered living materials. IMPORTANCE Engineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy-intensive processing or have limited tunability. We propose a bacterially synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step toward understanding the necessary parameters to engineering living cells to autonomously construct ELMs.

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1280: Increased Susceptibility to Genitovaginal Prolapse Associated with a Polymorphism in the Promoter of the Extracellular Matrix Protein LAMC1

The Journal of Urology ◽

10.1016/s0022-5347(18)31494-0 ◽

2007 ◽

Vol 177 (4S) ◽

pp. 421-422

Author(s):

Ganka Nikolova ◽

Christian O. Twiss ◽

Hane Lee ◽

Nelson Stanley ◽

Janet Sinsheimer ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Extracellular Matrix Protein ◽

Increased Susceptibility

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Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11438-0 ◽

2021 ◽

Author(s):

Aniel Moya-Torres ◽

Monika Gupta ◽

Fabian Heide ◽

Natalie Krahn ◽

Scott Legare ◽

...

Keyword(s):

Extracellular Matrix ◽

Hollow Fiber ◽

Mammalian Cells ◽

Matrix Protein ◽

Continuous Production ◽

Extracellular Matrix Protein ◽

Close Monitoring ◽

High Quality ◽

Biolayer Interferometry ◽

Hollow Fiber Bioreactor

Abstract The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. Key points • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality Graphical abstract

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