Atomic force microscopy of isolated mitochondria.

This chapter describes methods for isolating and imaging metabolically and toxicologically challenged mitochondria with atomic force microscopy. Mitochondria were isolated from rat dorsal root ganglia or brain and exposed to glucose or dinitrobenzene (DNB) to simulate the cellular environment of a diabetic animal that has been exposed to excess glucose or to DNB. It is one of only a few articles to present images of membrane structures, such as voltage-dependent, anion-selective channel pores, on intact organelles. The purpose of the chapter is not to report on the metabolic or toxic effects, but to communicate in more detail than a typical journal paper allows the methods used to image isolated organelles. We also provide a series images revealing the outer membrane and outer membrane pores. An image of an isolated nucleus as well as a set of notes written to avoid common pitfalls in isolation, labeling, and imaging is also included.

Dehydration-induced expression of a 31-kDa dehydrin in Polypodium polypodioides (Polypodiaceae) may enable large, reversible deformation of cell walls.

Current and predicted climate changes caused by global warming compel greater understanding of the molecular mechanisms that plants use to survive drought. The desiccation-tolerant fern Polypodium polypodioides exhibits extensive cell wall folding when dried to less than 15% relative water content (RWC) and rapidly (within 24 h) rehydrates when exposed to water and high humidity. A 31-kDa putative dehydrin polypeptide expressed in partially and fully dry tissues detected via western blotting was present only during drying and rapidly dissipated (within 24 h) upon tissue rehydration. Immunostaining indicates the presence of dehydrin near the cell wall of partially and fully dried tissues. Atomic force microscopy of tracheal scalariform perforations indicates that dry vascular tissue does not undergo significant strain. Additionally, environmental scanning electron microscopy reveals differential hydrophilicity between the abaxial and adaxial leaf surfaces as well as large, reversible deformation. The ability to avoid cell wall damage in some desiccation-tolerant species may be partially attributed to cell wall localization of dehydrins enabling reversible, large cell-wall deformation. Thus, the de novo synthesis of dehydrin proteins and potential localization to the cell walls of these desiccation-tolerant species may play a role in avoiding mechanical failure during drought.

Determination of the forces imposed by micro and nanopipettes during DOPC: DOPS liposome manipulation.

Using micropipette-based probing methods and an image processing algorithm for measuring deformation, the bending energies of aspirated DOPC:DOPS liposomes were estimated both before and during manipulation with an injection pipette. We found that unlike cells, which are penetrable with pipettes as large as 2mum in diameter and at speeds as slow as 4mum/s, liposomes, without a cytoskeleton to resist deformation, are impenetrable with pipettes as small as 25nm in diameter and at speeds as great as 4000mum/s. Using energy calculations and the previously published mechanical properties of DOPC:DOPS liposomes, the forces that injection pipettes of various sizes can exert onto liposomes during probing were estimated. Forces ranged from approximately 1pN to 6pN, and the forces exerted onto these liposomes increased as pipette size diminished. The quantification of the amount of force exerted on liposomes or cells during manipulation can assist in minimizing the damage during single-liposome, single-cell, or single-organelle injections and surgeries.

Parallel force measurement with a polymeric microbeam array using an optical microscope and micromanipulator.

An image analysis method and its validation are presented for tracking the displacements of parallel mechanical force sensors. Force is measured using a combination of beam theory, optical microscopy, and image analysis. The primary instrument is a calibrated polymeric microbeam array mounted on a micromanipulator with the intended purpose of measuring traction forces on cell cultures or cell arrays. One application is the testing of hypotheses involving cellular mechanotransduction mechanisms. An Otsu-based image analysis code calculates displacement and force on cellular or other soft structures by using edge detection and image subtraction on digitally captured optical microscopy images. Forces as small as 250+/-50 nN and as great as 25+/-2.5 microN may be applied and measured upon as few as one or as many as hundreds of structures in parallel. A validation of the method is provided by comparing results from a rigid glass surface and a compliant polymeric surface.

Cytoskeleton-membrane interactions in neuronal growth cones: a finite analysis study.

Revealing the molecular events of neuronal growth is critical to obtaining a deeper understanding of nervous system development, neural injury response, and neural tissue engineering. Central to this is the need to understand the mechanical interactions between the cytoskeleton and the cell membrane, and how these interactions affect the overall growth mechanics of neurons. Using finite element analysis, the stress in the membrane produced by an actin filament or a microtubule acting against a deformable membrane was modeled, and the deformation, stress, and strain were computed for the membrane. Parameters to represent the flexural rigidities of the well-studied actin and tubulin cytoskeletal proteins, as well as the mechanical properties of cell membranes, were used in the simulations. Our model predicts that a single actin filament is able to produce a normal contact stress on the cell membrane that is sufficient to cause membrane deformation but not growth. Our model also predicts that under clamped boundary conditions a filament with a buckling strength equal to or smaller than an actin filament would not cause the areal strain in the membrane to exceed 3%, and therefore the filament is incapable of causing membrane rupture or puncture to a safety factor of approximately 15-25. Decreasing the radius of the membrane upon which the normal contact stress is acting allows an increase in the amount of normal contact stress that the membrane can withstand before rupture. The model predicts that a 50 nm radius membrane can withstand approximately 4 MPa of normal contact stress before membrane rupture whereas a 250 nm radius membrane can withstand approximately 2.5 MPa. Understanding how the mechanical properties of cytoskeletal elements have coevolved with their respective cell membranes may yield insights into the events that gave rise to the sequences and superquaternary structures of the major cytoskeletal proteins. Additionally, numerical modeling of membranes can be used to analyze the forces and stresses generated by nanoscale biological probes during cellular injection.

Collagen's triglycine repeat number and phylogeny suggest an interdomain transfer event from a Devonian or Silurian organism into Trichodesmium erythraeum.

Two competing effects at two vastly different scales may explain collagen's current translation length. The necessity to have long molecules for maintaining mechanical integrity at the organism and supraorganism scales may be limited by the need to have small molecules capable of robust self-assembly at the nanoscale. The triglycine repeat regions of all 556 currently cataloged organisms with collagen-like genes were ranked by length. This revealed a sharp boundary in the GXY transcript number at 1032 amino acids (344 GXY repeats). An anomalous exception, however, is the intron-free Trichodesmium erythraeum collagen gene. Immunogold atomic force microscopy reveals, for the first time, the presence of a collagen-like protein in T. erythraeum. A phylogenetic protein sequence analysis which includes vertebrates, nonvertebrates, shrimp white spot syndrome virus, Streptococcus equi, and Bacillus cereus predicts that the collagen-like sequence may have emerged shortly after the divergence of fibrillar and nonfibrillar collagens. The presence of this anomalously long collagen gene within a prokaryote may represent an interdomain transfer from eukaryotes into prokaryotes that gives T. erythraeum the ability to form blooms that cover hundreds of square kilometers of ocean. We propose that the collagen gene entered the prokaryote intron-free only after it had been molded by years of mechanical selective pressure in larger organisms and only after large, dense food sources such as marine vertebrates became available. This anomalously long collagen-like sequence may explain T. erythraeum's ability to aggregate and thus concentrate its toxin for food-source procurement.

The effect of deformation on room temperature Coulomb blockade using conductive carbon nanotubes.

We report fluctuations in resistivity and the manifestation of Coulomb blockade phenomena of conductive multiwalled carbon nanotubes under buckling loads. Individual nanotubes were suspended and soldered between two indium-dipped tungsten probe tips. Using the electrical connection between the probes and the nanotube, electrical measurements were taken with the tube straight (unstrained) and bent (strained). Typical resistances were in the 10 G Omega range with resistivities in the 15 to 30 Omega-m range within the Coulomb blockade region of -1.0 to -0.4 V. Coulomb blockade, or electron tunneling events, appeared to occur at one of the contact points. This effect was diminished or lost once the carbon weld was broken.

Equal and local-load-sharing micromechanical models for collagens: quantitative comparisons in response of non-diabetic and diabetic rat tissue.

Chemical crosslinks in collagens resulting from binding of advanced glycation end-products, have long been presumed to alter the stiffness and permeability of glycated tissues. Recently, we developed a stochastic mechanical model for the response and failure of uniaxially deformed sciatic nerve tissue from diabetic and control rats. Here, we use our model to determine the likely correlation of fibril glycation with failure response, by quantifying statistical differences in their response. Our four-parameter model describes both the non-linear toe region and non-linear failure region of these tissues; the four parameters consist of (1) collagen fibril alignment, (2) fiber bundle waviness, (3) Weibull shape parameter for fibrillar strength, and (4) modulus-normalized Weibull scale parameter for fibrillar strength. Using an equal load sharing model we find that diabetic and control tissues had shape parameters of 9.88+/-5.50 and 4.33+/-3.67 (p=0.043), respectively, and scale parameters of 0.28+/-0.07 and 0.58+/-0.25 (p=0.033), respectively, implying that the diabetic tissue behaves in a more brittle manner, consistent with more highly crosslinked fibrils. We conclude that biochemical crosslinking directly affects measured mechanical properties. Further, this mechanical characterization may prove useful in mapping alterations in stiffness and permeability observed in glycated tissues.

Microfabrication procedure of PDMS microbeam array using photolithography for laminin printing and piconewton force transduction on axons.

The purpose of this paper is to introduce our design for transducing forces on the order of tens of piconewtons by optically measuring deflection of a microfabricated beam tip as it pulls on an array of flexible structures such as axons in an array of laminin-printed neurons. To achieve this we have designed polymeric beams with spring constants on the order of 10 pN/microm. We have fabricated circular microbeams with Sylgard polydimethylsiloxane (PDMS). The elastic modulus of PDMS was determined experimentally using a microscale and a micrometer at different concentrations of curing agent and base agent and found to be on the order of 100 kPa. The designed geometry is a 100x100 tapered microcone array with each beam having a length of 100 microm, and a base diameter of 10 microm. A SU-8 negative photoresist is etched using photolithography and used as a mold for PDMS soft lithography. PDMS was injected into the mold and the array peeled from the mold.

In situ imaging of mitochondrial outer-membrane pores using atomic force microscopy.

Here we describe a technique for imaging of the outer contours of the mitochondrial membrane using atomic force microscopy, subsequent to or during a toxic or metabolic challenge. Pore formation in both glucose-challenged and 1,3-dinitrobenzene (DNB)-challenged mitochondria was observed using this technique. Our approach enables quantification of individual mitochondrial membrane pore formations. With this work, we have produced some of the highest resolution images of the outer contours of the in situ mitochondrial membrane published to date. These are potentially the first images of the component protein clusters at the time of formation of the mitochondrial membrane transition pore in situ. With the current work, we have extended the application of atomic force microscopy of mitochondrial membranes to fluid imaging. We have also begun to correlate 3-D surface features of mitochondria dotted with open membrane pores with features previously viewed with electron microscopy (EM) of fixed sections.