Matrilins mediate weak cell attachment without promoting focal adhesion formation.

The matrilins form a family of non-collagenous adaptor proteins in the extracellular matrix. The extracellular ligand interactions of matrilins have been studied in some detail, while the potential interplay between matrilins and cells has been largely neglected. Except for matrilin-4, all matrilins mediate cell attachment, but only for matrilin-1 and -3 the binding is clearly dose dependent and seen already at moderate coating concentrations. Even so, much higher concentrations of matrilin-1 or -3 than of fibronectin are required for cell attachment to reach plateau values. Integrins contribute to the matrilin-mediated cell attachment, but the binding does not lead to formation of focal contacts and reorganisation of the actin cytoskeleton. Cells deficient in beta1 integrins are able to adhere, although weaker, and matrilins do not bind the soluble integrin alpha1beta1 and alpha2beta1 ectodomains. Cell surface proteoglycans may promote the attachment, as cells deficient in glycosaminoglycan biosynthesis adhere less well to matrilin-3. Even so, exogenous glycosaminoglycans are not able to compete for the attachment of HaCaT cells to matrilins.     

Dual color localization microscopy of cellular nanostructures

The dual color localization microscopy (2CLM) presented here is based on the principles of spectral precision distance microscopy (SPDM) with conventional autofluorescent proteins under special physical conditions. This technique allows us to measure the spatial distribution of single fluorescently labeled molecules in entire cells with an effective optical resolution comparable to macromolecular dimensions. Here, we describe the application of the 2CLM approach to the simultaneous nanoimaging of cellular structures using two fluorochrome types distinguished by different fluorescence emission wavelengths. The capabilities of 2CLM for studying the spatial organization of the genome in the mammalian cell nucleus are demonstrated for the relative distributions of two chromosomal proteins labeled with autofluorescent GFP and mRFP1 domains. The 2CLM images revealed quantitative information on their spatial relationships down to length-scales of 30 nm.     

Field-effect sensors with charged macromolecules: characterisation by capacitance-voltage, constant-capacitance, impedance spectroscopy and atomic-force microscopy methods

Field-effect-based capacitive electrolyte-insulator-semiconductor (EIS) sensors have been utilised for the deoxyribonucleic acid (DNA) immobilisation and hybridisation detection as well as for monitoring the layer-by-layer adsorption of polyelectrolytes (anionic poly(sodium 4-styrene sulfonate) (PSS) and cationic poly(allylamine hydrochloride) (PAH)). The EIS sensors with charged macromolecules have been systematically characterised by capacitance-voltage, constant-capacitance, impedance spectroscopy and atomic-force microscopy methods. The effect of the number and polarity of the polyelectrolyte layers on the shift of the capacitance-voltage curves has been investigated. Alternating potential shifts of about 30-90 mV have been observed after the adsorption of each polyanion and polycation layer, respectively. The DNA immobilisation and hybridisation signals were 35-55 and 24-33 mV, respectively. The possible mechanisms for the sensor responses are discussed.     

Control of mitochondrial and cellular respiration by oxygen

Control and regulation of mitochondrial and cellular respiration by oxygen is discussed with three aims: (1) A review of intracellular oxygen levels and gradients, particularly in heart, emphasizes the dominance of extracellular oxygen gradients. Intracellular oxygen pressure, $p_{O_2 } $ , is low, typically one to two orders of magnitude below incubation conditions used routinely for the study of respiratory control in isolated mitochondria. The $p_{O_2 } $ range of respiratory control by oxygen overlaps with cellular oxygen profiles, indicating the significance of $p_{O_2 } $ in actual metabolic regulation. (2) A methodologically detailed discussion of high-resolution respirometry is necessary for the controversial topic of respiratory control by oxygen, since the risk of methodological artefact is closely connected with far-reaching theoretical implications. Instrumental and analytical errors may mask effects of energetic state and partially explain the divergent views on the regulatory role of intracellular $p_{O_2 } $ . Oxygen pressure for half-maximum respiration,p ₅₀, in isolated mitochondria at state 4 was 0.025 kPa (0.2 Torr; 0.3 ΜM O₂), whereasp ₅₀ in endothelial cells was 0.06–0.08 kPa (0.5 Torr). (3) A model derived from the thermodynamics of irreversible processes was developed which quantitatively accounts for near-hyperbolic flux/ $p_{O_2 } $ relations in isolated mitochondria. The apparentp ₅₀ is a function of redox potential and protonmotive force. The protonmotive force collapses after uncoupling and consequently causes a decrease inp ₅₀. Whereas it is becoming accepted that flux control is shared by several enzymes, insufficient attention is paid to the notion of complementary kinetic and thermodynamic flux control mechanisms.     

Influence of Boron on the Membrane Potential in Elodea densa and Helianthus annuus Roots and H Extrusion of Suspension Cultured Daucus carota Cells

When following the membrane potential of Elodea densa leaf cells during a dark-light regime and analysing the different phases of the cycle, the pattern under boron deficiency resembled the one reported to occur after 3-(3,4-dichlorophenyl)-1,1-dimethylurea application. The potential in the dark slowly decreased when transferring Elodea densa leaflets and Helianthus annuus roots to a B-free medium and increased in the same way after B was added again. Addition of vanadate to inhibit plasmalemma ATPases in part mimicked the effects of B deficiency. It is suggested that B directly or indirectly affects the formation of a proton gradient. The effect of B on proton secretion was observed in various experiments with Daucus carota cell cultures. The results are discussed with respect to the possible involvement of B in membrane function and transport processes.     

The level of HER2 expression is a predictor of antibody-HER2 trafficking behavior in cancer cells

The receptor tyrosine kinase HER2 is known to play a central role in mitogenic signaling, motivating the development of targeted, HER2-specific therapies. However, despite the longstanding use of antibodies to target HER2, controversies remain concerning antibody/HER2 trafficking behavior in cancer cells. Understanding this behavior has direct relevance to the mechanism of action and effective design of such antibodies. In the current study, we analyzed the intracellular dynamics of trastuzumab, a marketed HER2-targeting antibody, in a panel of breast and prostate cancer cell lines that have a wide range of HER2 expression levels. Our results reveal distinct post-endocytic trafficking behavior of antibody-HER2 complexes in cells with different HER2 expression levels. In particular, HER2-overexpressing cells exhibit efficient HER2 recycling and limited reductions in HER2 levels upon antibody treatment, and consequently display a high level of antibody persistence on their plasma membrane. By contrast, in cells with low HER2 expression, trastuzumab treatment results in rapid antibody clearance from the plasma membrane combined with substantial decreases in HER2 levels and undetectable levels of recycling. A cell line with intermediate levels of HER2 expression exhibits both antibody recycling and clearance from the cell surface. Significantly, these analyses demonstrate that HER2 expression levels, rather than cell origin (breast or prostate), is a determinant of subcellular trafficking properties. Such studies have relevance to optimizing the design of antibodies to target HER2.     

Nonradioactive assay for detecting isoprenyl diphosphate synthase activity in crude plant extracts using liquid chromatography coupled with tandem mass spectrometry

Terpenoids form the largest class of plant metabolites involved in primary and secondary metabolism. Isoprenyl diphosphate synthases (IDSs) catalyze the condensation of the C(5) terpenoid building blocks, isopentenyl diphosphate and dimethylallyl diphosphate, to form geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)). These branch point reactions control the flow of metabolites that act as precursors to each of the major terpene classes-monoterpenes, sequiterpenes, and diterpenes, respectively. Thus accurate and easily performed assays of IDS enzyme activity are critical to increase our knowledge about the regulation of terpene biosynthesis. Here we describe a new and sensitive nonradioactive method for carrying out IDS assays using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to detect the short-chain prenyl diphosphate products directly without dephosphorylation. Furthermore, we were able to separate cisoid and transoid isomers of both C(10) enzyme products (geranyl diphosphate and neryl diphosphate) and three C(15) products [(E,E)-, (Z,E)-, and (Z,Z)-farnesyl diphosphate]. By applying the method to crude protein extracts from various organs of Arabidopsis thaliana, Nicotiana attenuata, Populus trichocarpa, and Picea abies, we could determine their IDS activity in a reproducible fashion.