Henry Shuman

Quantitative electron probe microanalysis of biological thin sections: Methods and validity

Abstract A quantitative method of electron probe X-ray analysis of thin sections proposed by Hall [1] and based on the relationship between characteristic peak and X-ray continuum was evaluated in conjunction with a multiple least squares computer fitting program designed for the subtraction of peak from background counts and for the deconvolution of overlapping peaks. The theory of this quantitation is discussed including the effects of higher Z elements in normally low Z biological matrices. Estimates of the inherent statistical errors of the deconvolution program and the extent of systematic errors introduced by changes in detector resolution and energy calibration are given. The minimal detectable concentration of K in a typical biological thin section and using probe currents readily tolerated by the specimen over a 100 sec counting time were found to be approximately 10 mmol/kg. The minimal detectable mass in a 100 sec collection time using a thermionic gun was calculated to be approximately 10 −19 g in agreement with previous analysis of the iron core of single ferritin molecules [7]. The quantitation of Na and K in thin polymer sections is illustrated and the use of biological materials containing covalently bound elements as standards is described. Quantitation of P and Mg of such specimens gave results within 10% of chemical analysis. The effects of the transmission function of the detection system, ionization cross section and fluorescence yield on the X-ray counts generated by different elements are evaluated by analyzing binary standards and fitting the calculated transmission function to the X-ray continuum. There was excellent agreement between the experimentally determined continuum shape, the calculated transmission function and the results obtained with binary standards. The continuum followed the behavior expected of thin organic sections up to 1 ∥micron. Above this thickness the absorption of the low energy continuum within the specimen was observed. The effects of contamination and mass loss due to radiation damage were evaluated by measuring X-ray continuum and characteristic peaks as a function of electron dose. Contamination was proportional to probe current and inversely proportional to probe diameter. The mass loss due to radiation damage, as measured by X-ray continuum counts, was significantly different in serum albumin and in sucrose specimens. There was a 50% loss of sucrose mass at a dose of 0.03 C/cm 2 at room temperature. An Arrhenius plot of the process gave a thermal activation energy of 900 K. We interpret this finding to indicate that the mass loss is primarily due to vaporization or diffusion of small organic fragments. Serum albumin films showed 13% mass loss at doses up to 760 C/cm 2 . The sources of extraneous, other than the specimen, continuum were evaluated and characterized. It is shown that such contributions to the continuum can be recognized as originating from a thick target X-ray spectrum (specimen grid and holder, electron optical column) or by a low energy profile that is inconsistent with the X-ray transmission function of the beryllium window of the detector (scattered electron spectrum). Methods for correcting for such extraneous contributions to the continuum are given, and the optimum region (lowest error specimen continuum) for quantitation is evaluated. Examples of quantitative analysis of cryosections of human red blood cells, frog striated and rabbit smooth muscle are given and shown to be, within the limits of statistical error, in agreement with the results of chemical analyses. The total theoretically attainable improvement in minimal mass detection is estimated to be a factor of approximately 300, suggesting a detectable minimal mass of 10 −22 g. We conclude that quantitative analysis of ultrathin biological sections with a spatial resolution of at least 2000 A and an absolute accuracy of approximately 10% is feasible with the method described.

Processive bidirectional motion of dynein-dynactin complexes in vitro

Cytoplasmic dynein is the primary molecular motor responsible for transport of vesicles, organelles, proteins and RNA cargoes from the periphery of the cell towards the nucleus along the microtubule cytoskeleton of eukaryotic cells. Dynactin, a large multi-subunit activator of dynein, docks cargo to the motor and may enhance dynein processivity. Here, we show that individual fluorescently labelled dynein–dynactin complexes exhibit bidirectional and processive motility towards both the plus and minus ends of microtubules. The dependence of this activity on substrate ATP concentration, nucleotide analogues and inhibitors suggests that bidirectional motility is an active energy-transduction property of dynein–dynactin motor mechano-chemistry. The unique motility characteristics observed may reflect the flexibility of the dynein structure that leads to an enhanced ability to navigate around obstacles in the cell.

Myosin I can act as a molecular force sensor.

The ability to sense molecular tension is crucial for a wide array of cellular processes, including the detection of auditory stimuli, control of cell shape, and internalization and transport of membranes. We show that myosin I, a motor protein that has been implicated in powering key steps in these processes, dramatically alters its motile properties in response to tension. We measured the displacement generated by single myosin I molecules, and we determined the actin-attachment kinetics with varying tensions using an optical trap. The rate of myosin I detachment from actin decreases >75-fold under tension of 2 piconewtons or less, resulting in myosin I transitioning from a low (<0.2) to a high (>0.9) duty-ratio motor. This impressive tension sensitivity supports a role for myosin I as a molecular force sensor.

Functional and Trafficking Defects in ATP Binding Cassette A3 Mutants Associated with Respiratory Distress Syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.

Binding strength and activation state of single fibrinogen-integrin pairs on living cells

Integrin activation states determine the ability of these receptors to mediate cell–matrix and cell–cell interactions. The prototypic example of this phenomenon is the platelet integrin, αIIbβ3. In unstimulated platelets, αIIbβ3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables αIIbβ3 to bind ligands such as fibrinogen and von Willebrand factor. To study the regulation of integrin activation states at the level of single molecules, we developed a model system based on laser tweezers, enabling us to determine the rupture forces required to separate single ligand-receptor pairs by using either purified proteins or intact living cells. Here, we show that rupture forces of individual fibrinogen molecules and either purified αIIbβ3 or αIIbβ3 on the surface of living platelets were 60 to 150 pN with a peak yield strength of 80–100 pN. Platelet stimulation using either ADP or the thrombin receptor-activating peptide enhanced the accessibility but not the adhesion strength of single αIIbβ3 molecules, indicating that there are only two states of αIIbβ3 activation. Thus, we found it possible to use laser tweezers to measure the regulation of forces between individual ligand-receptor pairs on living cells. This methodology can be applied to the study of other regulated cell membrane receptors using the ligand-receptor yield strength as a direct measure of receptor activation/inactivation state.

Electron energy loss analysis of near-trace-element concentrations of calcium.

The quantitation of near-trace-element concentrations of calcium (25 ppm atomic fraction) with electron energy loss spectroscopy (EELS) is demonstrated. The data collection, with an energy-stabilized parallel recording spectrometer, subsequent signal processing, and quantitation procedures are described. The quantitative results obtained with EELS, in the biologically relevant range of 1 to 100 mmol/kg, are directly compared with simultaneously collected and previously validated energy-dispersive X-ray spectroscopy (EPMA). The experimentally determined sensitivity of EELS for Ca detection is five-fold better than for EPMA, and the theoretically attainable sensitivity of EELS is ten-fold better than for EPMA. However, the attainment of this sensitivity with EELS is technically more difficult and limited by specimen thickness. The sensitivity of EELS experimentally demonstrated in this study permits the detection of three calcium atoms in a 10 nm diameter spot of an organic matrix, with a field-emission-gun-equipped scanning transmission electron microscope.

Kinesin and dynein-dynactin at intersecting microtubules: motor density affects dynein function.

Kinesin and cytoplasmic dynein are microtubule-based motor proteins that actively transport material throughout the cell. Microtubules can intersect at a variety of angles both near the nucleus and at the cell periphery, and the behavior of molecular motors at these intersections has implications for long-range transport efficiency and accuracy. To test motor function at microtubule intersections, crossovers were arranged in vitro using flow to orient successive layers of filaments. Single kinesin and cytoplasmic dynein-dynactin molecules fused with green-fluorescent protein, and artificial bead cargos decorated with multiple motors, were observed while they encountered intersections. Single kinesins tend to cross intersecting microtubules, whereas single dynein-dynactins have a more varied response. For bead cargos, kinesin motion is independent of motor number. Dynein beads with high motor numbers pause, but their actions become more varied as the motor number decreases. These results suggest that regulating the number of active dynein molecules could change a motile cargo into one that is anchored at an intersection, consistent with dyneins proposed transport and tethering functions in the cell.

Quantitative data processing of parallel recorded electron energy‐loss spectra with low signal to background

The performance of a multielement array detector for measuring small spectral features superimposed on a large background has been examined. In order to have the sensitivity of detection limited only by counting statistics, two factors have to be critically considered: the nonuniform spatial response of the array detector, and the possible addition of noise by the collection system. For the special case discussed here, energy‐loss spectroscopy of 100‐keV electrons, the added noise was small. The detective quantum efficiency (DQE) was measured to be DQE=0.87. Several techniques reducing the effect of the nonuniform response were tested: normalizing the spectra with an experimentally measured response curve, electronic differentiation to reduce the background, dynamic gain correction, and two methods of experimentally averaging the spatial response by measuring a sequence of repositioned spectra. Preservation of the statistical information present in a representative energy‐loss spectrum is shown to be feas...

ABCA3 Is Critical for Lamellar Body Biogenesis in Vivo

Mutations in ATP-binding cassette transporter A3 (human ABCA3) protein are associated with fatal respiratory distress syndrome in newborns. We therefore characterized mice with targeted disruption of the ABCA3 gene. Homozygous Abca3–/– knock-out mice died soon after birth, whereas most of the wild type, Abca3+/+, and heterozygous, Abca3+/–, neonates survived. The lungs from E18.5 and E19.5 Abca3–/– mice were less mature than wild type. Alveolar type 2 cells from Abca3–/– embryos contained no lamellar bodies, and expression of mature SP-B protein was disrupted when compared with the normal lung surfactant system of wild type embryos. Small structural and functional differences in the surfactant system were seen in adult Abca3+/– compared with Abca3+/+ mice. The heterozygotes had fewer lamellar bodies, and the incorporation of radiolabeled substrates into newly synthesized disaturated phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine in both lamellar bodies and surfactant was lower than in Abca3+/+ mouse lungs. In addition, since the fraction of near term Abca3–/– embryos was significantly lower than expected from Mendelian inheritance ABCA3 probably plays roles in development unrelated to surfactant. Collectively, these findings strongly suggest that ABCA3 is necessary for lamellar body biogenesis, surfactant protein-B processing, and lung development late in gestation.

Protein-protein unbinding induced by force: single-molecule studies.

Experiments in which two specifically interacting protein molecules are dissociated by external force have yielded new insights into mechanisms involved in cell adhesion, leukocyte rolling, regulation of integrin activity, antigen-antibody interactions and other protein-mediated reactions contingent upon molecular recognition. Another important aspect of force-induced protein-protein unbinding studies is the new information being gleaned about the thermodynamics and kinetics of bond rupture.