14 research outputs found
Imaging Molecular Structure of Channels and Receptors with an Atomic Force Microscope
Biological membranes contain specialized protein macromolecules such as channels, pumps and receptors. Physiologically, membranes and their constituent macromolecules are the interface surfaces toward which most of the regulatory biochemical and other signals are directed. Yet very little is known about these surfaces. The structure of biological membranes has been analyzed primarily using imaging techniques that are limited in their resolution of surface topology. An atomic force microscope (AFM) developed by Binnig, Quate and Gerber, can image molecular structures on specimen surfaces with subnanometer resolution, under diverse environmental conditions. Also, AFM can manipulate surfaces with molecular precision: it can nanodissect, translocate, and reorganize molecules on surfaces. The surface topology has been imaged for several hydrated channels, pumps and receptors which were a) present in isolated native membranes, b) reconstituted in artificial membrane or, c) expressed in an appropriate expression system. These images, at molecular resolution, reveal exciting new findings about their architecture. AFM induced force dissection reveals surfaces which are commonly inaccessible. In whole cell studies, in addition to the molecular structure of membrane receptors and channels, correlative electrical and biochemical activities have been examined. Such study suggests a single cell experiment where the structure-function correlation of many cloned channels and receptors can be understood
The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains
Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75% of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability
Gating of retinal transmission by afferent eye position and movement signals
Vision in most vertebrates is an active process that requires the brain to combine retinal signals with information about eye movement. Eye movement information may feed forward from the motor control areas of the brain or feed back from the extrinsic eye muscles. Feedback signals elicited by passive eye movement selectively gate retinal outflow at the first relay, the dorsal lateral geniculate nucleus. The gating predominantly facilitates retinogeniculate transmission immediately after eye movement and inhibits transmission when a new steady-state eye position is achieved. These two gating effects are distributed in a complementary fashion across the dorsal lateral geniculate nucleus such that the spatiotemporal activity profile could contribute to object detection and localization
Protegrin-1 (PG-1), An Antimicrobial Peptide Forms Ion Channels: Atomic Force Microscopy, Channel Conductance, and Molecular Dynamics Simulation Study
Atomic force microscopy and dissection of gap junctions
An atomic force microscope (AFM) was used to study the structure of isolated hepatic gap junctions in phosphate-buffered saline (PBS). The thickness of these gap junctions appears to be 14.4 nanometers, close to the dimensions reported by electron microscopy (EM). When an increasing force is applied to the microscope tip, the top membrane of the gap junction can be "dissected" away, leaving the extracellular domains of the bottom membrane exposed. When such "force dissection" is performed on samples both trypsinized and fixed with glutaraldehyde, the hexagonal array of gap junction hemichannels is revealed, with a center-to-center spacing of 9.1 nanometers
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Sub-nanowatt microfluidic single-cell calorimetry.
Non-invasive and label-free calorimetry could become a disruptive technique to study single cell metabolic heat production without altering the cell behavior, but it is currently limited by insufficient sensitivity. Here, we demonstrate microfluidic single-cell calorimetry with 0.2-nW sensitivity, representing more than ten-fold enhancement over previous record, which is enabled by (i) a low-noise thermometry platform with ultralow long-term (10-h) temperature noise (80 μK) and (ii) a microfluidic channel-in-vacuum design allowing cell flow and nutrient delivery while maintaining a low thermal conductance of 2.5 μW K-1. Using Tetrahymena thermophila as an example, we demonstrate on-chip single-cell calorimetry measurement with metabolic heat rates ranging from 1 to 4 nW, which are found to correlate well with the cell size. Finally, we perform real-time monitoring of metabolic rate stimulation by introducing a mitochondrial uncoupling agent to the microchannel, enabling determination of the spare respiratory capacity of the cells
Recommended from our members
Sub-nanowatt microfluidic single-cell calorimetry.
Non-invasive and label-free calorimetry could become a disruptive technique to study single cell metabolic heat production without altering the cell behavior, but it is currently limited by insufficient sensitivity. Here, we demonstrate microfluidic single-cell calorimetry with 0.2-nW sensitivity, representing more than ten-fold enhancement over previous record, which is enabled by (i) a low-noise thermometry platform with ultralow long-term (10-h) temperature noise (80 μK) and (ii) a microfluidic channel-in-vacuum design allowing cell flow and nutrient delivery while maintaining a low thermal conductance of 2.5 μW K-1. Using Tetrahymena thermophila as an example, we demonstrate on-chip single-cell calorimetry measurement with metabolic heat rates ranging from 1 to 4 nW, which are found to correlate well with the cell size. Finally, we perform real-time monitoring of metabolic rate stimulation by introducing a mitochondrial uncoupling agent to the microchannel, enabling determination of the spare respiratory capacity of the cells