Relaxation kinetics of biological dimer adsorption models

We discuss the relaxation kinetics of a one-dimensional dimer adsorption model as recently proposed for the binding of biological dimers like kinesin on microtubules. The non-equilibrium dynamics shows several regimes: irreversible adsorption on short time scales, an intermediate plateau followed by a power-law regime and finally exponential relaxation towards equilibrium. In all four regimes we give analytical solutions. The algebraic decay and the scaling behaviour can be explained by mapping onto a simple reaction-diffusion model. We show that there are several possibilities to define the autocorrelation function and that they all asymptotically show exponential decay, however with different time constants. Our findings remain valid if there is an attractive interaction between bound dimers.Comment: REVTeX, 6 pages, 5 figures; to appear in Europhys. Letters; a Java applet showing the simulation is accessible at http://www.ph.tum.de/~avilfan/rela

Modes of flagellar assembly in Chlamydomonas reinhardtii and Trypanosoma brucei

Defects in flagella growth are related to a number of human diseases. Central to flagellar growth is the organization of microtubules that polymerize from basal bodies to form the axoneme, which consists of hundreds of proteins. Flagella exist in all eukaryotic phyla, but neither the mechanism by which flagella grow nor the conservation of this process in evolution are known. Here, we study how protein complexes assemble onto the growing axoneme tip using (cryo) electron tomography. In Chlamydomonas reinhardtii microtubules and associated proteins are added simultaneously. However, in Trypanosoma brucei, disorganized arrays of microtubules are arranged into the axoneme structure by the later addition of preformed protein complexes. Post assembly, the T. brucei transition zone alters structure and its association with the central pair loosens. We conclude that there are multiple ways to form a flagellum and that species-specific structural knowledge is critical before evaluating flagellar defects

Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account

Axial Stretch of Rat Single Ventricular Cardiomyocytes Causes an Acute and Transient Increase in Ca(2+) Spark Rate

We investigate acute effects of axial stretch, applied by carbon fibers (CFs), on diastolic Ca(2+) spark rate in rat isolated cardiomyocytes. CFs were attached either to both cell ends (to maximize the stretched region), or to the center and one end of the cell (to compare responses in stretched and nonstretched half-cells). Sarcomere length was increased by 8.01 +/- 0.94% in the stretched cell fraction, and time series of XY confocal images were recorded to monitor diastolic Ca(2+) spark frequency and dynamics. Whole-cell stretch causes an acute increase of Ca(2+) spark rate (to 130.7 +/- 6.4%) within 5 seconds, followed by a return to near background levels (to 104.4 +/- 5.1%) within 1 minute of sustained distension. Spark rate increased only in the stretched cell region, without significant differences in spark amplitude, time to peak, and decay time constants of sparks in stretched and nonstretched areas. Block of stretch-activated ion channels (2 mu mol/L GsMTx-4), perfusion with Na(+)/Ca(2+)-free solution, and block of nitric oxide synthesis (1 mmol/L L-NAME) all had no effect on the stretch-induced acute increase in Ca(2+) spark rate. Conversely, interference with cytoskeletal integrity (2 hours of 10 mu mol/L colchicine) abolished the response. Subsequent electron microscopic tomography confirmed the close approximation of microtubules with the T-tubular-sarcoplasmic reticulum complex (to within approximate to 10(-8)m). In conclusion, axial stretch of rat cardiomyocytes acutely and transiently increases sarcoplasmic reticulum Ca(2+) spark rate via a mechanism that is independent of sarcolemmal stretch-activated ion channels, nitric oxide synthesis, or availability of extracellular calcium but that requires cytoskeletal integrity. The potential of microtubule-mediated modulation of ryanodine receptor function warrants further investigation. (Circ Res. 2009; 104: 787-795.

Processive Movement by a Kinesin Heterodimer with an Inactivating Mutation in One Head†

ABSTRACT: A single molecule of the motor enzyme kinesin-1 keeps a tight grip on its microtubule track, making tens or hundreds of discrete, unidirectional 8 nm steps before dissociating. This high duty ratio processive movement is thought to require a mechanism in which alternating stepping of the two head domains of the kinesin dimer is driven by alternating, overlapped cycles of ATP hydrolysis by the two heads. The R210K point mutation in Drosophila kinesin heavy chain was reported to disrupt the ability of the enzyme active site to catalyze ATP P-O bond cleavage. We expressed R210K homodimers as well as isolated R210K heads and confirmed that both are essentially inactive. We then coexpressed tagged R210K subunits with untagged wild-type subunits and affinity purified R210K/wild-type heterodimers together with the inactive R210K homodimers. In contrast to the R210K head or homodimer, the heterodimer was a highly active (>50 % of wild-type) microtubule-stimulated ATPase, and the heterodimer displayed high duty ratio processive movement in single-molecule motility experiments. Thus, dimerization of a subunit containing the inactivating mutation with a functional subunit can complement the mutation; this must occur either by lowering or by bypassing kinetic barriers in the ATPase or mechanical cycles of the mutant head. The observations provide support for kinesin-1 gating mechanisms in which one head stimulates the rate of essential processes in the other

Back on track – On the role of the microtubule for kinesin motility and cellular function

The evolution of cytoskeletal filaments (actin- and intermediate-filaments, and the microtubules) and their associated motor- and non-motor-proteins has enabled the eukaryotic cell to achieve complex organizational and structural tasks. This ability to control cellular transport processes and structures allowed for the development of such complex cellular organelles like cilia or flagella in single-cell organisms and made possible the development and differentiation of multi-cellular organisms with highly specialized, polarized cells. Also, the faithful segregation of large amounts of genetic information during cell division relies crucially on the reorganization and control of the cytoskeleton, making the cytoskeleton a key prerequisite for the development of highly complex genomes. Therefore, it is not surprising that the eukaryotic cell continuously invests considerable resources in the establishment, maintenance, modification and rearrangement of the cytoskeletal filaments and the regulation of its interaction with accessory proteins. Here we review the literature on the interaction between microtubules and motor-proteins of the kinesin-family. Our particular interest is the role of the microtubule in the regulation of kinesin motility and cellular function. After an introduction of the kinesin–microtubule interaction we focus on two interrelated aspects: (1) the active allosteric participation of the microtubule during the interaction with kinesins in general and (2) the possible regulatory role of post-translational modifications of the microtubule in the kinesin–microtubule interaction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42588/1/10974_2005_Article_9052.pd

Stress-induced in vivo recruitment of human cytotoxic natural killer cells favors subsets with distinct receptor profiles and associates with increased epinephrine levels

Acute stress drives a 'high-alert' response in the immune system. Psychoactive drugs induce distinct stress hormone profiles, offering a sought-after opportunity to dissect the in vivo immunological effects of acute stress in humans. 3,4-methylenedioxymethamphetamine (MDMA), methylphenidate (MPH), or both, were administered to healthy volunteers in a randomized, double-blind, placebo-controlled crossover-study. Lymphocyte subset frequencies, natural killer (NK) cell immune-phenotypes, and changes in effector function were assessed, and linked to stress hormone levels and expression of CD62L, CX3CR1, CD18, and stress hormone receptors on NK cells. MDMA/MPH > MDMA > MPH robustly induced an epinephrine-dominant stress response. Immunologically, rapid redistribution of peripheral blood lymphocyte-subsets towards phenotypically mature NK cells occurred. NK cytotoxicity was unaltered, but they expressed slightly reduced levels of the activating receptor NKG2D. Preferential circulation of mature NK cells was associated with high epinephrine receptor expression among this subset, as well as expression of integrin ligands previously linked to epinephrine-induced endothelial detachment. The acute epinephrine-induced stress response was characterized by rapid accumulation of mature and functional NK cells in the peripheral circulation. This is in line with studies using other acute stressors and supports the role of the acute stress response in rapidly mobilizing the innate immune system to counteract incoming threats

Low Temperature Specimen Preparation for Electron Tomography

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009</jats:p