Biomedical subjects

Arne Gennerich

Publications and source records attributed to Arne Gennerich.

6 recordsLinked to original sources

PubMed2006

Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3.

OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3-null mutation, suggesting that autoinhibition is important for OSM-3's biological function.

Adenosine Triphosphatases↗

PubMed2006

Single-molecule analysis of dynein processivity and stepping behavior.

Cytoplasmic dynein, the 1.2 MDa motor driving minus-end-directed motility, has been reported to move processively along microtubules, but its mechanism of motility remains poorly understood. Here, using S. cerevisiae to produce recombinant dynein with a chemically controlled dimerization switch, we show by structural and single-molecule analysis that processivity requires two dynein motor domains but not dynein's tail domain or any associated subunits. Dynein advances most frequently in 8 nm steps, although longer as well as side and backward steps are observed. Individual motor domains show a different stepping pattern, which is best explained by the two motor domains shuffling in an alternating manner between rear and forward positions. Our results suggest that cytoplasmic dynein moves processively through the coordination of its two motor domains, but its variable step size and direction suggest a considerable diffusional component to its step, which differs from Kinesin-1 and is more akin to myosin VI.

Amino Acid Sequence↗

PubMed2006

Finite-particle tracking reveals submicroscopic-size changes of mitochondria during transport in mitral cell dendrites.

The mechanisms of molecular motor regulation during bidirectional organelle transport are still uncertain. There is, for instance, the unsettled question of whether opposing motor proteins can be engaged in a tug-of-war. Clearly, any non-synchronous activation of the molecular motors of one cargo can principally lead to changes in the cargo's shape and size; the cargo's size and shape parameters would certainly be observables of such changes. We therefore set out to measure position, shape and size parameters of fluorescent mitochondria (during their transport) in dendrites of cultured neurons using a finite-particle tracking algorithm. Our data clearly show transport-related submicroscopic-size changes of mitochondria. The observed displacements of the mitochondrial front and rear ends are consistent with a model in which microtubule plus- and minus-end-directed motor proteins or motors of the same type but moving along anti-parallel microtubules are often out-of-phase and occasionally engaged in a tug-of-war. Mostly the leading and trailing ends of mitochondria undergo similar characteristic movements but with a substantial time delay between the displacements of both ends, a feature reminiscent of an inchworm-like motility mechanism. More generally, we demonstrate that observing the position, shape and size of actively transported finite objects such as mitochondria can yield information on organelle transport that is generally not accessible by tracking the organelles' centroid alone.

Animals↗

PubMed2004

Sizing-up finite fluorescent particles with nanometer-scale precision by convolution and correlation image analysis.

Determining the positions, shapes and sizes of finite living particles such as bacteria, mitochondria or vesicles is of interest in many biological processes. In fluorescence microscopy, algorithms that can simultaneously localize such particles as a function of time and determine the parameters of their shapes and sizes at the nanometer scale are not yet available. Here we develop two such algorithms based on convolution and correlation image analysis that take into account the position, orientation, shape and size of the object being tracked, and we compare the precision of the two algorithms using computer simulations. We show that the precision of both algorithms strongly depends on the object's size. In cases where the diameter of the object is larger than about four to five times the beam waist radius, the convolution algorithm gives a better precision than the correlation algorithm (it leads to more precise parameters), while for smaller object diameters, the correlation algorithm gives superior precision. We apply the convolution algorithm to sequences of confocal laser scanning micrographs of immobile Escherichia coli bacteria, and show that the centroid, the front end, the rear end, the left border and the right border of a bacterium can be determined with a signal-to-noise-dependent precision down to approximately 5 nm.

Algorithms↗

PubMed2003

Neuronal representation of odourants in the olfactory bulb of Xenopus laevis tadpoles.

When an odourant enters the nose, olfactory receptor neurons (ORNs) convey information about it to the olfactory bulb (OB), where this information is processed and where the first central representations of the odourant are generated. In this paper we show how odourants are represented by ensembles of OB neurons, in particular mitral cells (MCs) which are the output neurons of the OB. We were able to demonstrate for the first time that the intracellular calcium concentrations ([Ca2+]i) in the somata of these neurons undergo specific changes and that different stimuli are represented by different neuronal [Ca2+]i patterns. The similarity of patterns was assessed by cross-correlation analysis. We further show that noradrenaline (NA), which is reported to be involved in olfactory memory formation and to modulate synaptic transmission at dendrodendritic synapses in the OB, profoundly changes the representation of odourants at the level of MCs.

Animals↗

PubMed2002

Anisotropic diffusion in mitral cell dendrites revealed by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) can be used to measure kinetic properties of single molecules in drops of solution or in cells. Here we report on FCS measurements of tetramethylrhodamine (TMR)-dextran (10 kDa) in dendrites of cultured mitral cells of Xenopus laevis tadpoles. To interpret such measurements correctly, the plasma membrane as a boundary of diffusion has to be taken into account. We show that the fluorescence data recorded from dendrites are best described by a model of anisotropic diffusion. As compared to diffusion in water, diffusion of the 10-kDa TMR-dextran along the dendrite is slowed down by a factor 1.1-2.1, whereas diffusion in lateral direction is 10-100 times slower. The dense intradendritic network of microtubules oriented parallel to the dendrite is discussed as a possible basis for the observed anisotropy. In somata, diffusion was found to be isotropic in three dimensions and 1.2-2.6 times slower than in water.

Animals↗