Mechanics of the brain: perspectives,challenges, and opportunities

The human brain is the continuous subject of extensive investigation aimed at understanding its behavior and function. Despite a clear evidence that mechanical factors play an important role in regulating brain activity, current research efforts focus mainly on the biochemical or electrophysiological activity of the brain. Here, we show that classical mechanical concepts including deformations, stretch, strain, strain rate, pressure, and stress play a crucial role in modulating both brain form and brain function. This opinion piece synthesizes expertise in applied mathematics, solid and fluid mechanics, biomechanics, experimentation, material sciences, neuropathology, and neurosurgery to address today’s open questions at the forefront of neuromechanics. We critically review the current literature and discuss challenges related to neurodevelopment, cerebral edema, lissencephaly, polymicrogyria, hydrocephaly, craniectomy, spinal cord injury, tumor growth, traumatic brain injury, and shaken baby syndrome. The multi-disciplinary analysis of these various phenomena and pathologies presents new opportunities and suggests that mechanical modeling is a central tool to bridge the scales by synthesizing information from the molecular via the cellular and tissue all the way to the organ level.     

Closer proximity between opposing domains of vertebrate calmodulin following deletion of Met(145)-Lys(148)

To investigate the structural linkage between the opposing globular domains in vertebrate calmodulin (CaM), we have constructed a CaM mutant (CaMX(145)) deficient in the last four amino acids between Met(145) and Lys(148) at the carboxyl terminal. Circular dichroism and fluorescence spectroscopic measurements were used to detect changes in the average secondary and tertiary structure of CaMX(145) in comparison to full-length CaM. Complementary measurements of the maximal calcium-binding stoichiometry and ability to activate the plasma membrane (PM) Ca-ATPase permit an assessment of the functional significance of observed structural changes. In comparison with native CaM, we find that CaMX(145) exhibits (i) a large reduction in alpha-helical content, (ii) a dramatic decrease in the average spatial separation between the opposing globular domains, (iii) the loss of one high-affinity calcium-binding site, and (iv) a diminished binding affinity for the PM-Ca-ATPase. Thus, the sequence near the carboxyl terminus functions to stabilize high-affinity calcium binding at one site and facilitates important intramolecular interactions that maintain CaM in an extended conformation. However, despite the large conformational changes resulting from deletion of the last four amino acids at the carboxyl terminal, CaMX(145) can fully activate the PM-Ca-ATPase. These results indicate that target protein binding can restore the nativelike structure critical to function, emphasizing that the structure of the central helix is not critical to CaM function under equilibrium conditions. Rather, the central helix functions to maintain the spatial separation between the opposing domains in CaM that may be critical to high-affinity binding and the rapid activation of the PM-Ca-ATPase, which are necessary for optimal calcium signaling. Thus, following initial association between CaM and target proteins, structural changes involving the carboxyl-terminal sequence have the potential to play an important role in triggering the structural collapse of CaM that facilitates the rapid and cooperative binding of the opposing globular domains with target proteins, which is important to high-affinity binding and rapid enzyme activation.     

Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation

Phospholamban (PLB) associates with the Ca²⁺-ATPase in sarcoplasmic reticulum (SR) membranes to permit the modulation of contraction in response to -adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca²⁺-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, location, and turnover of endogenous and tagged proteins in myoblasts and during their differentiation. We found that PLB is constitutively expressed in both myoblasts and differentiated myotubes, whereas abundance increases of the Ca²⁺-ATPase coincide with the formation of differentiated myotubes. We observed that PLB is primarily present in highly mobile vesicular structures outside the endoplasmic reticulum, irrespective of the expression of the Ca²⁺-ATPase, indicating that PLB targeting is regulated through vesicle trafficking. Moreover, using pulse-chase methods, we observed that in myoblasts, PLB is trafficked through directed transport through the Golgi to the plasma membrane before endosome-mediated internalization. The observed trafficking of PLB to the plasma membrane suggests an important role for PLB during muscle differentiation, which is distinct from its previously recognized role in the regulation of the Ca²⁺-ATPase. sarco(endo)plasmic reticulum calcium-adenosine triphosphatase; differentiation; C2C12 myocytes; vesicle trafficking     

Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., Delta omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the Delta omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.     

Protein structure similarity from principle component correlation analysis

Background  

Owing to rapid expansion of protein structure databases in recent years, methods of structure comparison are becoming increasingly effective and important in revealing novel information on functional properties of proteins and their roles in the grand scheme of evolutionary biology. Currently, the structural similarity between two proteins is measured by the root-mean-square-deviation (RMSD) in their best-superimposed atomic coordinates. RMSD is the golden rule of measuring structural similarity when the structures are nearly identical; it, however, fails to detect the higher order topological similarities in proteins evolved into different shapes. We propose new algorithms for extracting geometrical invariants of proteins that can be effectively used to identify homologous protein structures or topologies in order to quantify both close and remote structural similarities.     

The inhibitory action of phospholamban involves stabilization of alpha-helices within the Ca-ATPase.

We have used attenuated total reflection Fourier transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopies to identify secondary and dynamic structural changes within the Ca-ATPase that result from the functional inhibition of transport activity by phospholamban (PLB). Isotopically labeled [(13)C]PLB was expressed and purified from Escherichia coli and was functionally reconstituted with unlabeled Ca-ATPase, permitting the resolution of the amide I and II absorbance bands of the Ca-ATPase from those of [(13)C]PLB. Upon co-reconstitution of the Ca-ATPase with PLB, spectral shifts are observed in both the CD spectra and the amide I and II bands associated with the Ca-ATPase, which are indicative of increased alpha-helical stability. Corresponding changes in the kinetics of H/D exchange occur upon association with PLB, indicating that 100 +/- 20 residues in the Ca-ATPase that normally undergo rapid amide H/D exchange become exchange resistant. There are no corresponding large changes in the secondary structure of PLB. The affinity of the structural interaction between PLB and the Ca-ATPase is virtually identical to that associated with functional inhibition (K(d) = 140 +/- 30 microM), confirming that the inhibitory regulation of the Ca-ATPase by PLB involves the stabilization of alpha-helices within the Ca-ATPase.     

A comparison between osmiophilic reagents and the Gomori lead method for the electron cytochemical demonstration of two lysosomal enzymes in oral epithelium

Synopsis Osmiophilic reagents were used to study the histochemical localization of acid phosphatase and non-specific esterase in the keratinized oral mucosa of rat. The reaction product from both enzymes was found in the epithelium and in cells of the corium as discrete granules, suggestive of a lysosomal localization. Treatment with E-600 before incubation for non-specific esterase did not change this localization. The osmium black end-product, due to acid phosphatase activity, was examined with the electron microscope and compared with the localization obtained by the Gomori lead phosphate technique. Both methods produced a reaction product in membrane-bounded bodies resembling lysosomes, as described in other tissues. These organelles were present in the basal prickle and granular cell layers of the epithelium. In the keratinized layer the reaction product was localized between the cell membranes of the deeper cells and no deposits were present in the cells. It is suggested that the osmiophilic reagents provide a good alternative to the Gomori method for the localization of lysosomal acid phosphatase at both the light and electron microscope levels.     

Calcium and lanthanide binding in the sarcoplasmic reticulum ATPase

The interactions of calcium and lathanides with the sarcoplasmic reticulum ATPase, and their respective ability to activate the enzyme, were studied by direct measurements of binding with radioactive tracers, functional effects on the ATPase partial reactions, changes in the quantum yield of tryptophanyl residues and a covalently bound fluorescein label (fluorescein 5-isothiocyanate, FITC), and energy transfer between bound lanthanide and fluorescent labels. We find that: (a) Lanthanides displace calcium from specific ATPase sites with diphasic kinetics that are consistent with sequential exchange. (b) Lanthanides in excess of the calcium stoichiometry are mostly bound to sarcoplasmic reticulum lipids and non-ATPase proteins. (c) Both calcium and lanthanides activate the ATPase and allow formation of the phosphorylated intermediate by utilization of ATP; however, hydrolytic cleavage of the intermediate formed in the presence of lanthanides occurs at a slower rate than the intermediate formed in the presence of calcium. (d) In contrast to a calcium-dependent change in the quantum yield of both the tryptophanyl residues (transmembrane region) and the FITC label (extramembranous region), lanthanides induce only a change in the quantum yield of the FITC label. (e) Measurements of energy transfer between bound lanthanide and fluorescent labels detect lanthanide bound midway between the catalytic site in the globular region of the ATPase outside the membrane, and the transmembrane calcium binding domain which is involved in enzyme activation (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989a) Nature 339, 476-478). It is apparent that cation bound in this midway location controls exchange of calcium bound in the transmembrane region. The possibility that the midway location may provide a domain for binding of a second calcium is discussed.     

Organization of the intercellular spaces of porcine epidermal and palatal stratum corneum: a quantitative study employing ruthenium tetroxide

Previous studies have demonstrated that the intercellular spaces of the stratum corneum contain multilamellar lipid sheets with variable ultrastructure in addition to desmosomes or desmosomal remnants. The intercellular lamellae are thought to provide a permeability barrier whereas the desmosomes are responsible for cell-cell cohesion. In this study, transmission electron microscopy of RuO₄-fixed tissue was used to compare the proportions of the intercellular spaces in epidermal and palatal stratum corneum occupied by desmosomes and by different patterns of lamellae. Desmosomes are more abundant in palatal than in epidermal stratum corneum (46.9 vs 15.0% length of intercellular space). In epidermis the most frequent lamellar arrangements involve 3 (23.5%) or 6 (24.2%) lucent bands with an alternating broad-narrow-broad pattern, whereas the most frequent lamellar arrangements in palatal tissue are 2 (17.2%) or 4 (10.5%) lucent bands of uniform width. Most of the nondesmosomal portion of the intercellular space in palatal stratum corneum was dilated and had elongated lamellae at the periphery and short disorganized lamellae and amorphous electron-dense material in the interior. It is concluded that the multilamellar lipid sheets are less extensive in palatal than in epidermal stratum corneum, which could explain the greater permeability of the palate.