Emrah Celik

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

Nonspecific interactions in AFM force spectroscopy measurements

Sample‐probe contact duration (dwell time) and loading force are two important parameters for the atomic force microscopy (AFM) force spectroscopy measurements of ligand–receptor interaction. A prolonged contact time may be required to initiate ligand–receptor binding as a result of slow on‐rate kinetics or low reactant density. In general, increasing contact duration promotes nonspecific interactions between the substrate and the functionalized cantilever and, thus, masking the detection of the specific interactions. To reduce the nonspecific interactions in AFM force measurements requiring extended substrate‐probe contact, we investigated the interaction of bovine serum albumin (BSA)‐functionalized cantilever with BSA‐coated glass, polyethylene glycol (PEG)‐functionalized glass, Pluronic‐treated Petri dishes and agarose beads. The frequency of nonspecific interaction between the BSA‐functionalized cantilever and the different samples increased with loading force and dwell time. This increase in nonspecific adhesion can be attributed to the interaction mediated by forced unfolding of BSA. By reducing the loading force, the contact duration of the AFM probe with an agarose bead can be extended to a few minutes without nonspecific adhesion. Copyright

Elongated membrane tethers, individually anchored by high affinity α4β1/VCAM-1 complexes, are the quantal units of monocyte arrests.

The α4β1 integrin facilitates both monocyte rolling and adhesion to the vascular endothelium and is physiologically activated by monocyte chemoattractant protein (MCP-1). The current study investigated the initial events in the adhesion of THP-1 cells to immobilized Vascular Cell Adhesion Molecule 1 (VCAM-1). Using AFM force measurements, cell adhesion was shown to be mediated by two populations of α4β1/VCAM-1 complexes. A low affinity form of α4β1 was anchored to the elastic elements of the cytoskeleton, while a higher affinity conformer was coupled to the viscous elements of the cell membrane. Within 100 ms of contact, THP-1 cells, stimulated by co-immobilized MCP-1, exhibited a tremendous increase in adhesion to VCAM-1. Enhanced cell adhesion was accompanied by a local decoupling of the cell membrane from the cytoskeleton and the formation of long membrane tethers. The tethers were individually anchored by multiple α4β1/VCAM-1 complexes that prolonged the extension of the viscous tethers. In vivo, the formation of these membrane tethers may provide the quantal structural units for the arrest of rolling monocytes within the blood vessels.

Experimental and Numerical Characterization of Non-Fickian Moisture Diffusion in Electronic Packages

This study investigates moisture diffusion characteristics of electronic packaging materials exhibiting Fickian and non-Fickian behavior. Experimental investigation involves moisture absorption and desorption tests of homogenous underfill materials and inhomogeneous organic substrates as examples of Fickian and non-Fickian solids, respectively. In absorption tests, samples are dried out in an oven prior to testing in a humid environmental chamber. In desorption tests, samples are saturated in the environmental chamber under a specified temperature and relative humidity prior to the moisture desorption inside the oven. Samples in both tests are taken out of the test environments and weighed frequently to obtain moisture weight change data. Using the measurements from testing several different Fickian and non-Fickian materials, diffusivity/moisture concentration relationships are constructed. These relationships are implemented into a customized finite element simulation tool under the ANSYSreg platform. Finally, the experimental tests on multi-material specimens are simulated by using this tool in order to establish its validity.

Effects of Nanoparticles on Stiffness and Impact Strength of Composites

The effects of nanoparticles on impact stiffness and strength are investigated. Nanoparticles are prepared from nanoclay particles embedded into carbon fiber/epoxy wet lay-up laminates. Specimens are characterized via low-velocity impact tests and following Cscanner damage analyses. The results show that the area of damage is reduced 46% when 5% nanoparticle is used in the composite. Modeling of the nanocomposites is performed by peridynamic simulations and compared with experimental measurements. The mode of damage and the improvement of the impact resistance of nanocomposites are captured successfully via numerical simulations.

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn(2+) additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1(-/-) neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn(2+)-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn(2+) induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.

Mechanical characterization of nickel nanowires by using a customized atomic force microscope.

A new experimental method to characterize the mechanical properties of metallic nanowires is introduced. An accurate and fast mechanical characterization of nanowires requires simultaneous imaging and testing of the nanowires. However, existing mechanical characterization techniques fail to accomplish this goal due either to the lack of imaging capability of the mechanical test setup or the difficulty of individual alignment and manipulation of single nanowires for each test. In this study, nanowire specimens prepared by an electroplating technique are located on a silicon substrate with trenches. A customized atomic force microscope is located inside a scanning electron microscope (SEM) in order to establish the visibility of the nanowires, and the tip of the atomic force microscope cantilever is utilized to bend and break the nanowires. The ability to visualize the nanowires in an SEM improves the speed and accuracy of the tests. Experimentally obtained force versus bending displacement curves are fitted into existing analytical formulations to extract the mechanical properties. Experimental results reveal that nickel nanowires have significantly higher strengths than their bulk counterparts, although their elastic modulus values are comparable to bulk nickel modulus values.

Rearrangement of microtubule network under biochemical and mechanical stimulations

Cells are constantly under the influence of various external forces in their physiological environment. These forces are countered by the viscoelastic properties of the cytoskeleton. To understand the response of the cytoskeleton to biochemical and mechanical stimuli, GFP-tubulin expressing CHO cells were investigated using scanning laser confocal microscopy. Cells treated with nocodazole revealed disruption in the microtubule network within minutes of treatment while keeping the cell shape intact. By contrast, trypsin, a proteolytic agent, altered the shape of CHO cells by breaking the peptide bonds at adhesion sites. CHO cells were also stimulated mechanically by applying an indentation force with an atomic force microscope (AFM) and by shear stress in a parallel plate flow chamber. Mechanical stimulation applied using AFM showed two distinct cytoskeletal responses to the applied force: an immediate response that resulted in the depolymerization and displacement of the microtubules out of the contact zone, and a slower response characterized by tubulin polymerization at the periphery of the indented area. Flow chamber experiments revealed that shear force did not induce formation of new microtubules in CHO cells and that detachment of adherent cells from the substrate occurred independent from the flow direction. Overall, the experimental system described here allows real-time characterization of dynamic changes in cell cytoskeleton in response to the mechano-chemical stimuli and, therefore, provides better understanding of the biophysical and functional properties of cells.

Scanning Ion Conductance Microscopic Study for Cellular Uptake of Cationic Conjugated Polymer Nanoparticles

Positively charged conjugated polymer nanoparticles (CPNs) are emerging biomaterials exhibiting high levels of cellular entry. High rate of cellular entry efficiency is believed that the amphiphilic CPNs interact efficiently with the negatively charged hydrophobic cellular membranes. For the first time, the cell surface morphological changes of human cervical cancer cells treated with CPNs using a scanning probe microscopy technique, scanning ion conductance microscopy (SICM) are imaged. After 1 h of CPN incubation, distinct changes are observed in cell surface morphology such as interconnected protrusions and pits with sub-micrometer sizes, which are not observed from cells treated with positively charged polyethyleneimine (PEI) under the same treatment conditions. The change on cell surface morphology is quantified by surface roughness ratio, which is increased as CPN concentration increases, while the ratio first increases and then decreases as the incubation time increases. These results suggest that cells respond actively toward CPN with both positive charges on the side chain and the hydrophobicity from rigid aromatic backbone, which leads to subsequent endocytosis. In conclusion, it is demonstrated that SICM is a suitable imaging technique to reveal the dynamic alternations on the cell surface morphology at the early stage of nanoparticles endocytosis with high resolution.

Metals Foams for Biomedical Applications: Processing and Mechanical Properties

Optimized structures found in nature can be sometimes imitated in engineering structures. The recent interest in functionally graded metallic materials makes bone structures interesting because bones are naturally functionally graded1. The cellular structure of foam metals (Fig.1) is very similar to that of the cancellous bone; therefore, these metals can be considered as potential candidates for future implant applications if porosity level, size and shape, strength and biocompatibility aspects satisfy the design specifications of implants. Foam metals based on biocompatible metallic materials (e.g. Ti and Ti-6A1-4V) are expected to provide better interaction with bone. This is mainly due to higher degree of bone growth into porous surfaces and higher degree of body fluid transport through three-dimensional interconnected array of pores2 (open cell foam), leading to better interlocking between implant and bone and hence reducing or avoiding the well-known implant losening. Furthermore, the elastic modulus of foam metals can be easily tailored with porosity level to match that of natural bone, leading to a better performance by avoiding the high degree of elastic mismatch which currently exists between conventional solid metallic implants and bone.