Douglas S. Martin

Cardiovascular responses to bicuculline in the paraventricular nucleus of the rat.

The present study was undertaken to determine whether y-aminobutyric acid in the paraventricular nucleus contributes to the regulation of cardiovascular function. Blood pressure and heart rate were recorded and plasma catecholamines were measured in conscious rats receiving microinfusions of either artificial cerebrospinal fluid or a y-aminobutyric acid antagonist, bicuculline methiodide, bilaterally into the paraventricular nucleus. Artificial cerebrospinal fluid had no effect on any of the recorded variables. In contrast, infusion of bicuculline into the region of the paraventricular nucleus produced increases in blood pressure (20 ±2 mm Hg), heart rate (110±ll beats/min), and plasma concentrations of norepinephrine (640±107 pg/ml) and epinephrine (lt266±267 pg/ml). Pretreatment with a ganglionic blocking agent abolished both the blood pressure (-1±2 mm Hg) and heart rate (5±18 beats/min) effects. Bilateral adrenal medullectomy reduced the changes in plasma norepinephrine concentrations (81 ±14 pg/ml) significantly and abolished the changes in plasma epinephrine concentrations (5±4 pg/ml). Conversely, adrenal medullectomy reduced the pressor effects (18±2 mm Hg) only slightly while the heart rate responses were attenuated (42 ±9 beats/min) by approximately 50%. These results suggest that an endogenous y-aminobutyric acid system exerts a tonic inhibitory effect on the sympathetic nervous system at the level of the paraventricular nucleus of the hypothalamus.

Apparent Subdiffusion Inherent to Single Particle Tracking

Subdiffusion and its causes in both in vivo and in vitro lipid membranes have become the focus of recent research. We report apparent subdiffusion, observed via single particle tracking (SPT), in a homogeneous system that only allows normal diffusion (a DMPC monolayer in the fluid state). The apparent subdiffusion arises from slight errors in finding the actual particle position due to noise inherent in all experimental SPT systems. A model is presented that corrects this artifact, and predicts the time scales after which the effect becomes negligible. The techniques and results presented in this paper should be of use in all SPT experiments studying normal and anomalous diffusion.

Measurement of diffusion in Langmuir monolayers by single-particle tracking

There is a great amount of literature available indicating that membranes are inhomogeneous, complex fluids. For instance, observation of diffusion in cell membranes demonstrated confined motion of membrane constituents and even subdiffusion. In order to circumvent the small dimensions of cells leading to weak statistics when investigating the diffusion properties of single membrane components, a technique based on optical microscopy employing Langmuir monolayers as membrane model systems has been developed in our lab. In earlier work, the motion of labeled single lipids was visualized. These measurements with long observation times, thus far only possible with this method, were combined with respective Monte-Carlo simulations. We could conclude that noise can lead in general to the assumption of subdiffusion while interpreting the results of single-particle-tracking (SPT) experiments within membranes in general. Since the packing density of lipids within monolayers at the air/water interface can be changed easily, inhomogeneity with regard to the phase state can be achieved by isothermal compression to coexistence regions. Surface charged polystyrene latexes were used as model proteins diffusing in inhomogeneous monolayers as biomembrane mimics. Epifluorescence microscopy coupled to SPT revealed that domain associated, dimensionally reduced diffusion can occur in these kinds of model systems. This was caused by an attractive potential generated by condensed domains within monolayers. Monte-Carlo simulations supported this view point. Moreover, long-time simulations show that diffusion coefficients of respective particles were dependent on the strength of the attractive potential present: a behavior reflecting altered dimensionality of diffusion. The widths of those potentials were also found to be affected by the domain size of the more ordered lipid phase. In biological membrane systems, cells could utilize these physical mechanisms to adjust diffusion properties of membrane components.

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and “limping.”

Curvature-induced expulsion of actomyosin bundles during cytokinetic ring contraction

Many eukaryotes assemble a ring-shaped actomyosin network that contracts to drive cytokinesis. Unlike actomyosin in sarcomeres, which cycles through contraction and relaxation, the cytokinetic ring disassembles during contraction through an unknown mechanism. Here we find in Schizosaccharomyces japonicus and Schizosaccharomyces pombe that, during actomyosin ring contraction, actin filaments associated with actomyosin rings are expelled as micron-scale bundles containing multiple actomyosin ring proteins. Using functional isolated actomyosin rings we show that expulsion of actin bundles does not require continuous presence of cytoplasm. Strikingly, mechanical compression of actomyosin rings results in expulsion of bundles predominantly at regions of high curvature. Our work unprecedentedly reveals that the increased curvature of the ring itself promotes its disassembly. It is likely that such a curvature-induced mechanism may operate in disassembly of other contractile networks. DOI: http://dx.doi.org/10.7554/eLife.21383.001

A fluorescent GTP analog as a specific, high-precision label of microtubules

Fluorescent imaging of cytoskeletal structures permits studies of both organization within the cell and dynamic reorganization of the cytoskeleton itself. Traditional fluorescent labels of microtubules, part of the cytoskeleton, have been used to study microtubule localization, structure, and dynamics, both in vivo and in vitro. However, shortcomings of existing labels make imaging of microtubules with high precision light microscopy difficult. In this paper, we report a new fluorescent labeling technique for microtubules, which involves a GTP analog modified with a bright, organic fluorophore (TAMRA, Cy3, or Cy5). This fluorescent GTP binds to a specific site, the exchangeable site, on tubulin in solution with a dissociation constant of 1.0±0.4 µM. Furthermore, the label becomes permanently incorporated into the microtubule lattice once tubulin polymerizes. We show that this label is usable as a single molecule fluorescence probe with nanometer precision and expect it to be useful for modern subdiffraction optical microscopy of microtubules and the cytoskeleton.

Measuring microtubule persistence length using a microtubule gliding assay.

The mechanical properties of microtubules have been an area of active research for the past two decades, in part because understanding the mechanics of individual microtubules contributes to modeling whole-cell rigidity and structure and hence to better understanding the processes underlying motility and transport. Moreover, the role of microtubule structure and microtubule-associated proteins (MAPs) in microtubule stiffness remains unclear. In this chapter, we present a kinesin-driven microtubule gliding assay analysis of persistence length that is amenable to simultaneous variation of microtubule parameters such as length, structure, or MAP coverage and determination of persistence length. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores, microtubule gliding trajectories are tracked with nanometer-level precision. The fluctuations in these trajectories, due to thermal fluctuations in the microtubules themselves, are analyzed to extract the microtubule persistence length. In the following, we describe this gliding assay and analysis and discuss two example microtubule variables, length and diameter, in anticipation that the method may be of wide use for in vitro study of microtubule mechanical properties.

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades(1). Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules(2-4), and even long microtubules have measured persistence lengths which vary by an order of magnitude(5-9). Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay(10). By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used(11). An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL. This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

Structural and Functional Analysis of Amyloid Fibril Formation by Two Closely Related Light Chains

Light chain amyloidosis (AL) is a hematological disorder in which a clonal population of B cells expands and secretes enormous amounts of immunoglobulin light chain protein. These light chains misfold and aggregate into amyloid fibrils, leading to organ dysfunction and death. In AL the sequence of the light chain is unique for each individual, giving rise to a highly variable course of disease. We are studying two proteins, designated AL09 and AL103, derived from the κI O18:O8 germline that are highly similar in sequence, yet had significant differences in the disease phenotype. We have studied the in vitro kinetics of fibril formation and the structure of the resulting fibrils in order to explain this phenomenon.We have begun by undertaking a systematic study of different solution properties and co-solutes that may affect fibril formation in these two proteins. We find that even though the proteins have similar thermodynamic properties, their fibril formation behavior is very different. AL09 readily forms fibrils under virtually every condition studied, while AL103 forms fibrils both more slowly and under fewer conditions. We have also explored the potential role of different glycosaminoglycans (GAGs) in the fibril formation process, specifically looking at the role of the size and charge of the GAG molecules. Furthermore, we have analyzed fibrils formed by these two disease proteins using limited proteolysis and mass spectrometry and have determined that in spite of their different phenotype the fibrils share a significant portion of their amyloid-forming core residues. Further structural studies are ongoing to determine how proteins with such different fibril formation kinetics can share a common amyloid structure.Funding by AHA SDG 06 3007N, NIH GM071514, and NIH F30DK082169