Dynamic interplay between breast cancer cells and normal endothelium mediates the expression of matrix macromolecules,proteasome activity and functional properties of endothelial cells

Background

Breast cancer–endothelium interactions provide regulatory signals facilitating tumor progression. The endothelial cells have so far been mainly viewed in the context of tumor perfusion and relatively little is known regarding the effects of such paracrine interactions on the expression of extracellular matrix (ECM), proteasome activity and properties of endothelial cells.

Methods

To address the effects of breast cancer cell (BCC) lines MDA-MB-231 and MCF-7 on the endothelial cells, two cell culture models were utilized; one involves endothelial cell culture in the presence of BCCs-derived conditioned media (CM) and the other co-culture of both cell populations in a Transwell system. Real-time PCR was utilized to evaluate gene expression, an immunofluorescence assay for proteasome activity, and functional assays (migration, adhesion and invasion) and immunofluorescence microscopy for cell integrity and properties.

Results

BCC-CM decreases the cell migration of HUVEC. Adhesion and invasion of BCCs are favored by HUVEC and HUVEC-CM. HA levels and the expression of CD44 and HA synthase-2 by HUVEC are substantially upregulated in both cell culture approaches. Adhesion molecules, ICAM-1 and VCAM-1, are also highly upregulated, whereas MT1-MMP and MMP-2 expressions are significantly downregulated in both culture systems. Notably, the expression and activity of the proteasome β5 subunit are increased, especially by the action of MDA-MB-231-CM on HUVEC.

Conclusions and general significance

BCCs significantly alter the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells. Deep understanding of such paracrine interactions will help to design novel drugs targeting breast cancer at the ECM level. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Sialic acids attached to N- and O-glycans within the Nav1.4 D1S5–S6 linker contribute to channel gating

Background

Voltage-gated Na⁺ channels (Na_v) are responsible for the initiation and conduction of neuronal and muscle action potentials. Na_v gating can be altered by sialic acids attached to channel N-glycans, typically through isoform-specific electrostatic mechanisms.

Methods

Using two sets of Chinese Hamster Ovary cell lines with varying abilities to glycosylate glycoproteins, we show for the first time that sialic acids attached to O-glycans and N-glycans within the Na_v1.4 D1S5–S6 linker modulate Na_v gating.

Results

All measured steady-state and kinetic parameters were shifted to more depolarized potentials under conditions of essentially no sialylation. When sialylation of only N-glycans or of only O-glycans was prevented, the observed voltage-dependent parameter values were intermediate between those observed under full versus no sialylation. Immunoblot gel shift analyses support the biophysical data.

Conclusions

The data indicate that sialic acids attached to both N- and O-glycans residing within the Na_v1.4 D1S5-S6 linker modulate channel gating through electrostatic mechanisms, with the relative contribution of sialic acids attached to N- versus O-glycans on channel gating being similar.

General significance

Protein N- and O-glycosylation can modulate ion channel gating simultaneously. These data also suggest that environmental, metabolic, and/or congenital changes in glycosylation that impact sugar substrate levels, could lead, potentially, to changes in Na_v sialylation and gating that would modulate AP waveforms and conduction.     

Endothelial cells in culture: An experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [³H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor- and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selectin And vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also rn rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.Abbreviations AGEs advanced glycated end-products - ICAM intracellular adhesion molecule - IL-1 interleukin-1 - IFN- interferon- - MECIF monocyte-derived endothelial cell inhibitory factor - NO nitric oxide - PECAM-1 platelet-endothelial cell adhesion molecule-1 - RAGE receptor for advanced glycated end-products - RBCs red blood cells - TNF- tumor necrosis factor- - VCAM vascular adhesion molecule     

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

Background

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.

Methods

Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.

Results

Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.

Conclusions

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.

General significance

This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.     

Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

Background

In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown.

Results

To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2.

Conclusions

These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.     

Characterization of the in vitro binding and inhibition kinetics of primary amine oxidase/vascular adhesion protein-1 by glucosamine

Background

Primary-amine oxidase (PrAO) catalyzes the oxidative deamination of endogenous and exogenous primary amines and also functions, in some tissues, as an inflammation-inducible endothelial factor, known as vascular adhesion protein-1. VAP-1 mediates the slow rolling and adhesion of lymphocytes to endothelial cells in a number of inflammatory conditions, including inflammation of the synovium.

Methods

Glucosamine binding to the enzyme was assessed spectrofluorometrically and the kinetics of inhibition of PrAO were determined spectrophotometrically through the use of direct or coupled assays, in the presence of different substrates.

Results

Glucosamine is not a substrate for PrAO, but acts as a time-dependent inhibitor of PrAO activity, displaying mixed inhibition kinetics. The observed inhibition and binding were augmented in the presence of H₂O₂.

Conclusions

Significant in vitro effects on PrAO require glucosamine in the millimolar concentration range and it is not clear at this stage whether a low but persistent level of PrAO inhibition might contribute to the anti-arthritic response.

General significance

This work was aimed at characterizing the interactions of PrAO/VAP-1 with glucosamine, a widely used “over-the-counter” supplement for the treatment of osteoarthritis.     

Dicer knockdown induces fibronectin-1 expression in HEK293T cells via induction of Egr1

Background

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs); depletion of Dicer was found to impair the migration of endothelial cells.

Methods

siRNA transfection, cell migration assay, real-time RT–PCR, chromatin immunoprecipitation, Western blotting, ELISA, caspase-3 activity assay, and annexin-V–FITC assay were utilized.

Results

Knockdown of Dicer impairs the migratory capacity of HEK293T cells and induces fibronectin-1. The upregulation of fibronectin-1 is dependent on Egr1. Fibronectin-1/Dicer double-knockdown cells showed a marked increase in apoptosis compared with fibronectin-1 single knockdown cells.

Conclusions

Decreased Dicer expression induces fibronectin-1 expression via an Egr1-dependent manner.

General significance

Our data suggest that upregulation of fibronectin-1 protects Dicer knockdown HEK293T cells against apoptosis.     

Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents

Background

Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur.

Methods

Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone.

Results

Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde–dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes.

Conclusions and general significance

Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents.     

Investigation of red blood cell mechanical properties using AFM indentation and coarse-grained particle method

Background

Red blood cells (RBCs) deform significantly and repeatedly when passing through narrow capillaries and delivering dioxygen throughout the body. Deformability of RBCs is a key characteristic, largely governed by the mechanical properties of the cell membrane. This study investigated RBC mechanical properties using atomic force microscopy (AFM) with the aim to develop a coarse-grained particle method model to study for the first time RBC indentation in both 2D and 3D. This new model has the potential to be applied to further investigate the local deformability of RBCs, with accurate control over adhesion, probe geometry and position of applied force.

Results

The model considers the linear stretch capacity of the cytoskeleton, bending resistance and areal incompressibility of the bilayer, and volumetric incompressibility of the internal fluid. The model’s performance was validated against force–deformation experiments performed on RBCs under spherical AFM indentation. The model was then used to investigate the mechanisms which absorbed energy through the indentation stroke, and the impact of varying stiffness coefficients on the measured deformability. This study found the membrane’s bending stiffness was most influential in controlling RBC physical behaviour for indentations of up to 200 nm.

Conclusions

As the bilayer provides bending resistance, this infers that structural changes within the bilayer are responsible for the deformability changes experienced by deteriorating RBCs. The numerical model presented here established a foundation for future investigations into changes within the membrane that cause differences in stiffness between healthy and deteriorating RBCs, which have already been measured experimentally with AFM.

     

The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs

Background

The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs).

Methods

Deep partial thickness burn model in Sprague–Dawley rats and the heat denatured cell model (52 °C, 35 s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin.

Results

Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs.

Conclusions

The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF.

General significance

We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis.     

Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease

Background

The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties.

Methods

Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL.

Results

ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption.

Conclusions

ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis.

General significance

The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.     

Syndecan-2 regulation of morphology in breast carcinoma cells is dependent on RhoGTPases

Background

While syndecan-2 is usually considered a mesenchymal transmembrane proteoglycan, it can be upregulated in some tumour cells, such as the malignant breast carcinoma cell line, MDA-MB231. Depletion of this syndecan by siRNA, but not other syndecans, has a marked effect on cell morphology, increasing spreading, microfilament bundle and focal adhesion formation, with reduced cell migration.

Methods

A combination of siRNA transfection, immunofluorescence microscopy, phosphoprotein analysis and migration assays was used to determine how syndecan-2 may influence the cytoskeleton.

Results

The altered adhesion upon syndecan-2 depletion was dependent on the RhoGTPases. p190ARhoGAP relocated to the margins of spreading cells, where it codistributed with syndecan-4 and active β₁-integrin. This was accompanied by increased RhoGAP tyrosine phosphorylation, indicative of activity and RhoGTPase suppression. Consistent with this, GTP-RhoA was strongly present at the edges of control cells, but lost after syndecan-2 reduction by siRNA treatments. Further, RhoA, but not RhoC was shown to be essential for the anchored phenotype of these breast carcinoma cells that accompanied siRNA-mediated loss of syndecan-2.

Conclusions

Syndecan-2 has a key role in promoting the invasive activity of these cells, in part by regulating the RhoGTPases.

General significance

Syndecan-2, as a cell surface receptor is accessible for targeting to determine whether breast tumour progression is altered. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Sialosignaling: Sialyltransferases as engines of self-fueling loops in cancer progression

Background

Glycosylation is increasingly recognized as one of the most relevant postranslational modifications. Sialic acids are negatively charged sugars which frequently terminate the carbohydrate chains of glycoproteins and glycolipids. The addition of sialic acids is mediated by sialyltransferases, a family of glycosyltransferases with a crucial role in cancer progression.

Scope of the review

To describe the phenotypic and clinical implications of altered expression of sialyltransferases and of their cognate sialylated structures in cancer. To propose a unifying model of the role of sialyltransferases and sialylated structures on cancer progression.

Major conclusions

We first discuss the biosynthesis and the role played by the major cancer-associated sialylated structures, including Thomsen–Friedenreich-associated antigens, sialyl Lewis antigens, α2,6-sialylated lactosamine, polysialic acid and gangliosides. Then, we show that altered sialyltransferase expression in cancer, consequence of genetic and epigenetic alterations, generates a flow of information toward the membrane through the biosynthesis of aberrantly sialylated molecules (inside-out signaling). In turn, the presence of aberrantly sialylated structures on cell membrane receptors generates a flow of information toward the nucleus, which can exacerbate the neoplastic phenotype (outside-in signaling). We provide examples of self-fueling loops generated by these flows of information.

General significance

Sialyltransferases have a wide impact on the biology of cancer and can be the target of innovative therapies. Our unified view provides a conceptual framework to understand the impact of altered glycosylation in cancer.     

Protective protein/cathepsin A rescues N-glycosylation defects in neuraminidase-1

Background

Neuraminidase-1 (NEU1) catabolizes the hydrolysis of sialic acids from sialo-glycoconjugates. NEU1 depends on its interaction with the protective protein/cathepsin A (PPCA) for lysosomal compartmentalization and catalytic activation. Murine NEU1 contains 4 N-glycosylation sites, 3 of which are conserved in the human enzyme. The expression of NEU1 gives rise to differentially glycosylated proteins.

Methods

We generated single-point mutations in mouse NEU1 at each of the 4 N-glycosylation sites. Mutant enzymes were expressed in NEU1-deficient cells in the presence and absence of PPCA.

Results

All 4 N-glycosylation variants were targeted to the lysosomal/endosomal compartment. All N-glycans, with the exception of the most C-terminal glycan, were important for maintaining stability or catalytic activity. The loss of catalytic activity caused by the deletion of the second N-glycan was rescued by increasing PPCA expression. Similar results were obtained with a human NEU1 N-glycosylation mutant identified in a sialidosis patient. The N-terminal N-glycan of NEU1 is indispensable for its function, whereas the C-terminal N-glycan appears to be non-essential. The omission of the second N-glycan can be compensated for by upregulating the expression of PPCA.

General significance

These findings could be relevant for the design of target therapies for patients carrying specific NEU1 mutations.     

Endothelium-dependent epithelial–mesenchymal transition of tumor cells: Exclusive roles of transforming growth factor β1 and β2

Background

Induction of epithelial–mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not.

Methods

Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor β (TGFβ) antibodies were used to neutralize TGFβ₁, TGFβ₂ and TGFβ₃. Expression and release of TGFβ proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively.

Results

Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFβ₁ and TGFβ₂ proteins and much lower level of TGFβ₃ protein. Conditioned medium from these endothelial cells contained TGFβ₁ and TGFβ₂, but TGFβ₃ could not be detected. Neutralizing antibody against each of TGFβ₁ or TGFβ₂ did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFβ₁ and TGFβ₂ completely abolished it.

Conclusions

Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFβ₁ and TGFβ₂.

General significance

The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium.     

Tryptophan end-tagging of antimicrobial peptides for increased potency against Pseudomonas aeruginosa

Background

Due to increasing antibiotics resistance, antimicrobial peptides (AMPs) are receiving increased attention. Pseudomonas aeruginosa is a major pathogen in this context, involved, e.g., in keratitis and wound infections. Novel bactericidal agents against this pathogen are therefore needed.

Methods

Bactericidal potency was monitored by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was probed by hemolysis. Mechanistic information was obtained from assays on peptide-induced vesicle disruption and lipopolysaccharide binding.

Results

End-tagging by hydrophobic amino acids yields increased potency of AMPs against P. aeruginosa, irrespective of bacterial proteinase production. Exemplifying this by two peptides from kininogen, GKHKNKGKKNGKHNGWK and KNKGKKNGKH, potency increased with tag length, correlating to more efficient bacterial wall and vesicle rupture, and to more pronounced P. aeruginosa lipopolysaccharide binding. End-tag effects remained at high electrolyte concentration and in the presence of plasma or anionic macromolecular scavengers. The tagged peptides displayed stability against P. aeruginosa elastase, and were potent ex vivo, both in a contact lens model and in a skin wound model.

General significance

End-tagging, without need for post-peptide synthesis modification, may be employed to enhance AMP potency against P. aeruginosa at maintained limited toxicity.     

Förster Resonance Energy Transfer — An approach to visualize the spatiotemporal regulation of macromolecular complex formation and compartmentalized cell signaling

Background

Signaling messengers and effector proteins provide an orchestrated molecular machinery to relay extracellular signals to the inside of cells and thereby facilitate distinct cellular behaviors. Formations of intracellular macromolecular complexes and segregation of signaling cascades dynamically regulate the flow of a biological process.

Scope of review

In this review, we provide an overview of the development and application of FRET technology in monitoring cyclic nucleotide-dependent signalings and protein complexes associated with these signalings in real time and space with brief mention of other important signaling messengers and effector proteins involved in compartmentalized signaling.

Major conclusions

The preciseness, rapidity and specificity of cellular responses indicate restricted alterations of signaling messengers, particularly in subcellular compartments rather than globally. Not only the physical confinement and selective depletion, but also the intra- and inter-molecular interactions of signaling effectors modulate the direction of signal transduction in a compartmentalized fashion. To understand the finer details of various intracellular signaling cascades and crosstalk between proteins and other effectors, it is important to visualize these processes in live cells. Förster Resonance Energy Transfer (FRET) has been established as a useful tool to do this, even with its inherent limitations.

General significance

FRET technology remains as an effective tool for unraveling the complex organization and distribution of various endogenous signaling proteins, as well as the spatiotemporal dynamics of second messengers inside a single cell to distinguish the heterogeneity of cell signaling under normal physiological conditions and during pathological events.     

Photodamage of lipid bilayers by irradiation of a fluorescently labeled cell-penetrating peptide

Background

Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl–CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process.

Methods

In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles.

Results

The peptide TAT labeled with 5(6)-carboxytetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence.

Conclusions

These results suggest that the cell-penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently.

General significance

Understanding the mechanism behind Fl–CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds.     

Quantifying fibronectin adhesion with nanoscale spatial resolution on glycosaminoglycan doped polypyrrole using Atomic Force Microscopy

Background

The interaction of ECM proteins is critical in determining the performance of materials used in biomedical applications such as tissue regeneration, implantable bionics and biosensing.

Methods

To improve our understanding of ECM protein–conducting polymer interactions, we have used Atomic Force Microscopy (AFM) to elucidate the interactions of fibronectin (FN) on polypyrrole (PPy) doped with different glycosaminoglycans.

Results

We were able to classify four main types of FN interactions, including those related to 1) non-specific adhesion, 2) protein unfolding and subsequent unbinding from the surface, 3) desorption and 4) interactions with no adhesion. FN adhesion on PPy/hyaluronic acid showed a significantly lower density of surface adhesion with the adhesion restricted to nodule structures, as opposed to their peripheries, of the polymer morphology. In contrast, PPy/chondroitin sulfate showed a significantly higher density of surface adhesion to the point where the distribution of adhesion effectively masked the topography. Through conductive AFM imaging, we found that the conductive regions correlated with regions of FN adhesion.

Conclusions

Given that the conductivity requires doping of the polymer, these findings suggest that FN adhesion is mediated by interactions with chondroitin sulfate and hyaluronic acid at the polymer surface and may be indicative of specific interactions due to contributions from electrostatic attraction between the FN and sulfate/anionic groups of the dopants.

General significance

This study demonstrates the ability of AFM to resolve the protein–conducting polymer interactions at the molecular and nanoscale level, which will be important for interfacing these polymer materials with biological systems. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

Role of Macromolecular Depletion in Red Blood Cell Adhesion

The adhesion of red blood cells (RBCs) to cells or surfaces is of current basic science and clinical interest yet the details governing this process are still being explored. In this study, the effects of nonadsorbing polymers on the adhesion of RBC to albumin-coated glass have been investigated employing interference reflection microscopy. Our experimental results indicate that adhesion can be induced in the presence of dextran with a molecular mass ≥70 kDa and that the induced forces are strong enough to significantly suppress membrane undulations. The overall dependence of the adhesion energies on the polymer concentration is consistent with the assumption that macromolecular depletion induces this attractive interaction. In conclusion, our results indicate that depletion interaction might play a significant role in RBC adhesion via initiating close contacts, and thus suggest the importance of depletion forces for RBC interactions and its relevance to a wide variety of in vitro and in vivo cell-cell and cell-surface interactions.