Diatom adhesive mucilage contains distinct supramolecular assemblies of a single modular protein

A previous study used atomic force microscopy saw-tooth retraction curves to characterize the adhesive mucilage pads of the diatom Toxarium undulatum. The major mucilage component consisted of adhesive nanofibers (ANFs) made up of modular proteins arranged into cohesive units, each containing a set number of modular proteins aligned in parallel. This study shows that T. undulatum adhesive mucilage is a biocomposite containing four additional adhesive components, including single modular proteins that are likely to be the structural units from which the ANFs are assembled. Two further distinct supramolecular assemblies were observed to coexist with ANFs (ANFs II and III), along with a continuum of single modular proteins through oligomers made up of varying numbers of modular proteins arranged in parallel. All components of the adhesive biocomposite produce a characteristic force spectrum with the same interpeak distance (35.3 +/- 0.3 (mean +/- SE) nm), suggesting they are derived from discrete supramolecular assemblies of the same modular protein, but they are distinguishable from one another based on the rupture force, persistence length, and interpeak force measured from their saw-tooth curves.     

Adhesive modular proteins occur in the extracellular mucilage of the motile, pennate diatom Phaeodactylum tricornutum

This Letter reports on adhesive modular proteins recorded by atomic force microscopy on live cells from the extracellular mucilage secreted from, and deposited around, the motile form of the pennate diatom Phaeodactylum tricornutum. This is the first report of modular proteins and their supramolecular assemblies, called adhesive nanofibers (ANFs), to be found on diatoms that use adhesives not only for substratum adhesion, but as a conduit for cell motility. The permanent adhesive pads secreted by Toxarium undulatum, a sessile centric diatom, were previously shown to possess ANFs with a modular protein backbone. Our results reported here suggest that modular proteins may be an important component of diatom adhesives in general, and that diatoms utilize the tensile strength, toughness, and flexibility of ANFs for multiple functions. Significantly, the genome of P. tricornutum has recently been sequenced; this will allow directed searches of the genome to be made for genes with modular protein homologs, and subsequent detailed studies of their molecular structure and function.     

Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy

Type IV pili play an important role in bacterial adhesion, motility, and biofilm formation. Here we present high-resolution atomic force microscopy (AFM) images of type IV pili from Pseudomonas aeruginosa bacteria. An individual pilus ranges in length from 0.5 to 7 microm and has a diameter from 4 to 6 nm, although often, pili bundles in which the individual filaments differed in both length and diameter were seen. By attaching bacteria to AFM tips, it was possible to fasten the bacteria to mica surfaces by pili tethers. Force spectra of tethered pili gave rupture forces of 95 pN. The slopes of force curves close to the rupture force were nearly linear but showed little variation with pilus length. Furthermore, force curves could not be fitted with wormlike-chain polymer stretch models when using realistic persistence lengths for pili. The observation that the slopes near rupture did not depend on the pili length suggests that they do not represent elastic properties of the pili. It is possible that this region of the force curves is determined by an elastic element that is part of the bacterial wall, although further experiments are needed to confirm this.     

Characterization of the adhesive mucilages secreted by live diatom cells using atomic force microscopy

Atomic Force Microscopy (AFM) resolved the topography and mechanical properties of two distinct adhesive mucilages secreted by the marine, fouling diatom Craspedostauros australis. Tapping mode images of live cells revealed a soft and cohesive outer mucilage layer that encased most of the diatom's siliceous wall, and force curves revealed an adhesive force of 3.58 nN. High loading force, contact mode imaging resulted in cantilever 'cleaned' cell walls, which enabled the first direct observation of the active secretion of soft mucilage via pore openings. A second adhesive mucilage consisted of strands secreted at the raphe, a distinct slit in the silica wall involved in cell-substratum attachment and motility. Force measurements revealed a raphe adhesive strand(s) resistant to breaking forces up to 60 nN, and these strands could only be detached from the AFM cantilever probe using the manual stepper motor.     

Elasticity and adhesion of resting and lipopolysaccharide-stimulated macrophages

Colloidal Force Microscopy was employed to study the viscoelastic and adhesive properties of macrophages upon stimulation with lipopolysaccharide (LPS). Force vs. distance measurements were performed. The adhesion of LPS-stimulated cells (separation force=37+/-3 nN) was almost twice as high as that of resting macrophages (16+/-1 nN). Upon retraction pulling of membrane tethers was observed. Tether lengths and forces at which rupture take place did not depend on stimulation. The reduced Young's modulus K, a measure of cytoskeleton elasticity, was three times lower than that of the control. The data show that LPS has profound effects on cytomechanical and adhesion properties of macrophages.     

Assessing the flexibility of intermediate filaments by atomic force microscopy

Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.     

Mechanical properties of desiccated ragweed pollen grains determined by micromanipulation and theoretical modelling

The mechanical properties of desiccated ragweed pollen grains were determined using a micromanipulation technique and a theoretical model. Single pollen grains with a diameter of approximately 20 microm were compressed and held, compressed and released, and compressed to rupture at different speeds between two parallel surfaces. Simultaneously, the force being imposed on the pollen grains was measured. It has been found that the rupture force of pollen grains increased linearly with their displacement at rupture on average, but was independent of their diameter. The mean rupture force was 1.20 +/- 0.03 mN, and mean deformation (the ratio between the displacement and diameter) at rupture was 22 +/- 0.6%. Single pollen grains were modeled as a capsule with a core full of air and a non permeable wall. A constitutive equation based on Hookean law was used to determine the mechanical property parameters Eh (product of the Young's modulus and wall thickness), and the mean value of Eh of desiccated pollen gains was estimated to be 1653 +/- 36 N/m.     

Discrete interactions in cell adhesion measured by single-molecule force spectroscopy

Cell-cell adhesion mediated by specific cell-surface molecules is essential for multicellular development. Here we quantify de-adhesion forces at the resolution of individual cell-adhesion molecules, by controlling the interactions between single cells and combining single-molecule force spectroscopy with genetic manipulation. Our measurements are focused on a glycoprotein, contact site A (csA), as a prototype of cell-adhesion proteins. csA is expressed in aggregating cells of Dictyostelium discoideum, which are engaged in development of a multicellular organism. Adhesion between two adjacent cell surfaces involves discrete interactions characterized by an unbinding force of 23 +/- 8 pN, measured at a rupture rate of 2.5 +/- 0.5 microm s-1.     

Adhesion molecules from the diatom Phaeodactylum tricornutum (Bacillariophyceae): genomic identification by amino‐acid profiling and in vivo analysis

Cell adhesion molecules (CAMs) are important in prokaryotes and eukaryotes for cell–cell and cell–substratum interactions. The characteristics of adhesive proteins in the model diatom Phaeodactylum tricornutum were investigated by bioinformatic analysis and in vivo characterization. Bioinformatic analysis of the protein coding potential of the P. tricornutum genome used an amino‐acid profile that we developed as a new system to identify uncharacterized or novel CAMs. Putative diatom CAMs were identified and seven were characterized in vivo, by generation of transgenic diatom lines overexpressing genes encoding C‐terminal yellow fluorescent protein (YFP) fusion proteins. Three of these selected genes encode proteins with weak similarity to characterized proteins, a c‐type lectin and two fasciclins, whereas the others are novel. The resultant cell lines were investigated for alterations in their adhesive ability. Whole cell‐substratum adhesion strength was measured in a fully turbulent flow chamber, while atomic force microscopy was used to quantify the relative frequency of adhesion, as well as the length and strength of single molecules in the secreted mucilage. Finally, quartz crystal microbalance analysis characterized the visco‐elastic properties and interaction of the mucilage–substratum interface. These combined studies revealed a range of phenotypes affecting adhesion, and led to the identification of candidate proteins involved in diatom adhesion. In summary, our study has for the first time combined bioinformatics and molecular physiological studies to provide new insights into diatom adhesive molecules.     

Force measurements on myelin basic protein adsorbed to mica and lipid bilayer surfaces done with the atomic force microscope.

The mechanical and adhesion properties of myelin basic protein (MBP) are important for its function, namely the compaction of the myelin sheath. To get more information about these properties we used atomic force microscopy to study tip-sample interaction of mica and mixed dioleoylphosphatidylserine (DOPS) (20%)/egg phosphatidylcholine (EPC) (80%) lipid bilayer surfaces in the absence and presence of bovine MBP. On mica or DOPS/EPC bilayers a short-range repulsive force (decay length 1.0-1.3 nm) was observed during the approach. The presence of MBP always led to an attractive force between tip and sample. When retracting the tip again, force curves on mica and on lipid layers were different. While attached to the mica surface, the MBP molecules exhibited elastic stretching behavior that agreed with the worm-like chain model, yielding a persistence length of 0.5 +/- 0.25 nm and an average contour length of 53 +/- 19 nm. MBP attached to a lipid bilayer did not show elastic stretching behavior. This shows that the protein adopts a different conformation when in contact with lipids. The lipid bilayer is strongly modified by MBP attachment, indicating formation of MBP-lipid complexes and possibly disruption of the original bilayer structure.     

Effects of hypoxia on pulmonary microvascular volume

To determine the effects of alveolar hypoxia on pulmonary microvascular volume, X-ray microfocal angiographic images of isolated perfused dog lung lobes were obtained during passage of a bolus of radiopaque contrast medium during both normoxic (alveolar gas, 15% O(2), 6% CO(2), and 79% N(2)) and hypoxic (3% O(2), 6% CO(2), and 91% N(2)) conditions. Regions of interest (ROIs) over the lobar artery and vein at low magnification and a feeding artery ( approximately 500 microm diameter) and the nearby microvasculature (vessels smaller than approximately 50 microm) at high magnification were identified, and X-ray absorbance vs. time curves were acquired under both conditions from the same ROIs. The total pulmonary vascular volume was calculated from the flow and the mean transit time for the contrast medium passage from the lobar artery to lobar vein. The fractional changes in microvascular volume were determined from the areas under the high-magnification X-ray absorbance curves. Hypoxia decreased lobar volume by 13 +/- 3% (SE) and regional microvascular volume by 26 +/- 4% (SE). Given the morphometry of the lung vasculature, these results suggest that capillary volume was decreased by hypoxia.     

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes)

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.     

Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.     

Length-tension relationships of small arteries, veins, and lymphatics from the rat mesenteric microcirculation

The passive and active length-tension relationships of isolated rat mesenteric lymphatics ( approximately 150 microm ID), and adjacent small arteries ( approximately 240 microm) and veins ( approximately 275 microm) were compared under isometric conditions using a wire myograph. About 60% of the lymphatic vessels developed spontaneous contractions in physiological saline solution at nominal preload. To maximally activate smooth muscle, 145 mM K(+) + 5 x 10(-5) M norepinephrine was used for arteries, and 145 mM K(+) + 1 x 10(-6) M substance P was used for lymphatics and veins. In response, arteries exhibited monotonic force development to a plateau level, whereas lymphatics and veins showed biphasic force development, consisting of a transient force peak followed by partial relaxation to a plateau over approximately 5 min. The passive and the active length-tension curves were similar in shape among all three vessels. However, the maximal active tension of arteries (3.4 +/- 0.42 mN/mm) was significantly greater than peak active tension (0.59 +/- 0.04 mN/mm) or plateau tension (0.20 +/- 0.04 mN/mm) in small veins and greater than peak active tension (0.34 +/- 0.02 mN/mm) or plateau tension (0.21 +/- 0.02 mN/mm) in lymphatics. Maximal active medial wall stress was similar between lymphatics and veins but was approximately fivefold higher in small arteries. For lymphatics, the pressure calculated from the optimal preload was significantly higher than that found previously in isobaric studies of isolated lymphatics, suggesting the capacity to operate at higher than normal pressures for increased responsiveness. Our results represent the first mechanical comparisons of arterial, venous, and lymphatic vessels in the same vasculature.     

Selective adhesion and mineral deposition by osteoblasts on carbon nanofiber patterns

In an effort to develop better orthopedic implants, osteoblast (bone-forming cells) adhesion was determined on microscale patterns (30 microm lines) of carbon nanofibers placed on polymer substrates. Patterns of carbon nanofibers (CNFs) on a model polymer (polycarbonate urethane [PCU]) were developed using an imprinting method that placed CNFs in selected regions. Results showed the selective adhesion and alignment of osteoblasts on CNF patterns placed on PCU. Results also showed greater attraction forces between fibronectin and CNF (compared with PCU) patterns using atomic force microscope force-displacement curves. Because fibronectin is a protein that mediates osteoblast adhesion, these results provide a mechanism of why osteoblast adhesion was directed towards CNF patterns. Lastly, this study showed that the directed osteoblast adhesion on CNF patterns translated to enhanced calcium phosphate mineral deposition along linear patterns of CNFs on PCU. Since CNFs are conductive materials, this study formulated substrates that through electrical stimulation could be used in future investigations to further promote osteoblasts to deposit anisotropic patterns of calcium-containing mineral similar to that observed in long bones.     

A live bioprobe for studying diatom-surface interactions

Atomic force microscopy has been employed to compare the adhesion of Navicula species I diatoms to surfaces of a hydrophobic elastomer, Intersleek, and a hydrophilic mineral, mica. This was accomplished using tipless atomic force microscopy cantilevers functionalized with live diatom cells. Both surfaces were tested with the same diatom bioprobe. Force versus distance curves generated during these experiments revealed comparable cell adhesion strengths on Intersleek and mica, indicating that Navicula diatoms secrete extracellular polymeric substances with hydrophobic and hydrophilic properties. A statistical analysis of force curves was carried out and the average values of works of detachment of a diatom from Intersleek and mica surfaces were determined.     

How to develop globular proteins into adhesives

To make globular proteins suitable for application in adhesives, the specific bonds and interactions which shape their structure have to broken. Only then, a layer of relatively large, flexible and interwoven polymer chains, which are firmly attached to the solid surface by adsorption, can be created. Such a network layer is essential to save the adhesive bond under an applied force, because it can distribute the concentration of stresses generated at the interface into the bulk. Unfolding and swelling of a protein can be achieved by changing the solvent quality. For the globular whey protein beta-lactoglobulin, the optimal conditions for unfolding and swelling is found with 98% formic acid as a solvent. In formic acid, beta-lactoglobulin looses its amphoteric character (it is protonated, probably for approximately 20%). In addition, formic acid is less polar than water and thus a better solvent for the apolar parts of the protein. The swelling and unfolding behaviour of beta-lactoglobulin is studied by viscosity and CD-spectroscopy measurements. For the interpretation of the results we apply the Kuhn formalism that the conformation of a protein can be described in terms of a statistical chain which consists of segments of an average persistence length P. The statistical segment length P, which varies with the experimental conditions, is directly related to the adsorption energy required for a strong adhesion between coil and surface. It determines the depletion energy kT P(-2) m(-2) which must be overcome by specific attraction between side groups of the protein chain and the surface. For beta-lactoglobulin in 98% formic acid, we find a P value of approximately 2.2 nm, pointing at a relatively flexible chain. The minimum net adsorption energy kT P(-2) is then approximately 1 mJ m(-2), a relatively small value to be exceeded. Preliminary results of destructive adhesion tests on beech wood lap-shear joints reveal promising tensile strengths of approximately 2.9+/-1.1 N mm(-2), indeed.     

Cell-substratum adhesion strength as a determinant of hepatocyte aggregate morphology

Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary-spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415-426, 1997.     

Synthetic peptides that mimic the adhesive recognition signal of fibronectin: differential effects on cell-cell and cell-substratum adhesion in embryonic chick cells

Although fibronectin has been implicated in cell-cell as well as cell-substratum interactions, most experimentation has focused on cell-substratum interactions of fibroblasts. We have examined the effect of the specific peptide GRGDS derived from the cell-binding sequence of fibronectin upon cell-cell and cell-substratum interactions using embryonic cells and tissues. Embryonic chick segmental plate cells undergo compaction (i.e., increased cell-cell adhesion) during the early stages of somitogenesis. Fibronectin has been implicated in this increase in cell-cell interaction. In contrast, precardiac mesoderm undergoes directional migration upon a fibronectin-rich substratum, exhibiting both cell-cell and cell-substratum interactions. The segmental plate cells, which are the precursors of embryonic somites, normally show very little cell-cell or cell-substratum interaction in culture. These cells exhibit a striking increase in intercellular adhesion, but exhibit no cell-substratum adhesion, in the presence of relatively low concentrations of the fibronectin-derived peptide GRGDS. Somite cells, which normally exhibit both cell-cell and cell-substratum adhesion in culture, show complete inhibition of cell-substratum adhesion in the presence of this peptide. Precardiac mesoderm, which normally exhibits both cell-cell and cell-substratum adhesion in culture, shows a marked inhibition of both processes in the presence of GRGDS. Since the finding that a monovalent competitive inhibitor of fibronectin binding can stimulate cell-cell adhesion was unexpected, we propose a "trigger" hypothesis, whereby the peptide recognition signal acts as a specific signal or trigger for the morphogenetic process of compaction. There is a striking specificity to this effect, since synthetic peptides with even conservative changes in the amino acid sequence have no effect. Finally, we find that under certain conditions the effect of the specific peptide is lost in 6-8 hr and the cells resume cell-substratum interactions or, in the case of the segmental plate cells, revert from the compacted state and exhibit a substantial decrease in cell-cell adhesion. Our studies indicate the diversity of cell and tissue responses possible when even a single peptide inhibitor of adhesion, and we have identified the first known activating effect of a fibronectin peptide on cell behavior and differentiation.     

THE STRUCTURE AND NANOMECHANICAL PROPERTIES OF THE ADHESIVE MUCILAGE THAT MEDIATES DIATOM‐SUBSTRATUM ADHESION AND MOTILITY1

We investigated the adhesive mucilage and mechanism of cell‐substratum adhesion of two benthic raphid diatoms, the marine species Craspedostauros australis E. J. Cox and the freshwater species Pinnularia viridis (Nitzsch) Ehrenberg. SEM images of P. viridis and C. australis cells revealed the presence of multistranded tethers that appear to arise along the raphe openings and extend for a considerable distance from the cell before forming a “holdfast‐like” attachment with the substratum. We propose that the tethers result from the elongation/stretching of composite adhesive mucilage strands secreted from raphes during the onset of cell adhesion and reorientation. Atomic force microscopy (AFM) force measurements reveal that the adhesive strands originating from the nondriving raphe of live C. australis and P. viridis are highly extensible and accumulate to form tethers. During force measurements tethers can be chemically stained and are seen to extend between the cantilever tip and a cell during elongation and relaxation. In most cases, AFM force measurements recorded an interaction with a number of adhesive strands that are secreted from the raphe. The force curves of C. australis and P. viridis revealed a sawtooth pattern, suggesting the successive unbinding of modular domains when the adhesive strands were placed under stress. In addition, we applied the “fly‐fishing” technique that allowed the cantilever, suspended a distance above the cell, to interact with single adhesive strands protruding from the raphe. These force curves revealed sawtooth patterns, although the binding forces recorded were in the range for single molecule interactions.