Experimental Biologists Meet Theoreticians

Abstracts


Uri Ashery, Tel Aviv University
Vesicle mobility and exocytosis

Synaptic transmission involves the fusion of presynaptic vesicles and the release of neurotransmitter to the synaptic cleft. Before fusion vesicles have to undergo a priming step, which renders them fusion competent. To learn about the nature of the priming process and to characterize the proteins that are involved in this step we use total internal reflection fluorescent microscopy (TIRF). Using TIRF we can characterize vesicle mobility, turnover and release probability under different conditions. I will present data showing that tomosyn, a synaptic protein that inhibits exocytosis, causes a reduction in the pool of immobile vesicles, the same pool from which exocytosis preferentially takes place and increases vesicle turnover rate. Possible implication will be discussed.

*****

Michael Elbaum, Weizmann Institute of Science
Nucleocytoplasmic transport: a reversible mechanism

The nucleus is a defining organelle in the cells of higher organisms. As the genes are stored within the nucleus while the encoded protein expression takes place in the cytoplasm, these cells require biochemical machinery for molecular traffic between the two major compartments. The nuclear pore complex fulfills this need. More than simply a gateway, it acts as a molecular pump. We have investigated the mechanism and capacity of this pump using cell-free nuclei reconstituted in vitro in Xenopus egg extract. Surprisingly, we observe that directional accumulation takes place in the presence of a bidirectional flux across the pore, i.e. without vectorial translocation. A far-reaching consequence is that the nuclear localization signal dictates the fate of a protein population rather than the individual molecule, which remains free to shuttle back and forth. This implies an open communication between the nucleus and cytoplasm, and a ubiquitous mechanism for signaling in both directions.

*****

David Holcman, Weizmann Institute of Science
Modeling molecular trafficking in the cytoplasm

Cellular functions rely on chemical reactions occurring in microdomains which may involve a few amount of molecules. In recent years, various approaches have emerged to analyze cell function at a molecular level. In this talk, I will review some recent theoretical approach to study molecular trafficking in microdomains. These methods are based on analysis related to the mean first passage time of a random molecule to a small hole located on the boundary of a domain. The talk is built around two examples: First, the synaptic weight depends on the type and the number of receptors it contains. Because receptors can move by lateral diffusion on the surface of neurons, the receptor number fluctuates; this raises the question of synapses stability. The second example concerns the trafficking of organelles, DNA, molecules inside the cell cytoplasm and the mean time to travel from the cell surface to the nucleus.

*****

Baruch Minke, Hebrew University of Jerusalem
Open channel block by Ca2+ underlies light adaptation and voltage dependence of Drosophila TRP and TRPL channels

Invertebrate photoreceptors are sensitive to absorption of single photons and yet function efficiently during intense lights by adjusting their sensitivity in a wide range of light intensities via a still unclear Ca2+-dependent adaptation mechanism. Here we show using single channel analysis and whole cell recordings that the light activated channels of Drosophila, TRP and TRP-like (TRPL) are major target of Ca2+ dependent adaptation. We furthermore show that open channel block by Ca2+ underlies this unknown mechanism of light adaptation and the hitherto unexplained voltage dependence of the TRP and TRPL channels. The main evidence for open channel block and its role in light adaptation are: i. The outward rectification of the expressed TRPL channels arise from removal of Ca2+ block by depolarization, thereby increasing the opening frequency of channel openings. ii. The duration of channel openings is strongly Ca2+ dependent. iii. The decline of the light induced current during illumination, the hallmark of light adaptation, depends on the number of open channels, on membrane voltage and on Ca2+ level. iv. Positive membrane voltage, which relieves open channel block by Ca2+ antagonize light adaptation. v. The divalent cations Ba2+ and Mg2+ which induce open channel block on the channels also induce light adaptation. The vast amount of TRP and TRPL channels, which is by far larger than needed to reach saturated light response, is therefore required by an open channel block mechanism to maintain the wide dynamic range of light adaptation.

*****

Avi Minsky, Weizmann Institute of Science
Physical and structural aspects of DNA repair

*****

Jacob Rubinstein, Technion and Indiana University
Transport in thin networks

In many applications one needs to study transport processes in a thin network. For example we mention heat flow in long narrow tubes, mesoscopic superconducting devices, fluid flow in the coronary network, hydro-elastic waves in the cochlea, etc. A useful approach for such problems is to derive simpler models in which the transport takes place on a graph that is the geometrical limit of the network. Some of these simpler models are fairly obvious, while others are not. I shall survey a number of such limit models taken from a variety of applications.

*****

Zeev Schuss, Tel Aviv University
What can be calculated for particles diffusing in a small domain?

The dependence on the diffusion coefficient and on geometric features of the first passage time of a diffuser to a small absorber can be predicted analytically or numerically. Numerical simulations of transitions through narrow channels can be speeded up by calculating the statistics of the first passage time. The diffusive motion of shaped particles, e.g. DNA, can be modeled and analyzed by coupled stochastic differential equations. The diffusion of charged and shaped particles near dielectric boundaries can be described by effective anisotropic diffusion equations. The interaction between external magnetic radiation and the gating charge in a voltage gated channel can be described in terms of stochastic resonance.

*****

Menahem Segal and Eduard Korkotian, Weizmann Institute of Science
Local diffusion of calcium ions in a small sphere within neurons

Dendritic spines are unique calcium compartments, independent of their parent dendrite; elevation of [Ca2+]i induced by synaptic activity or by local photolysis of caged calcium is typically confined to the spine head, less than 1┬Ám3 in volume. Thus, the spine neck has been proposed to constitute a critical regulator of calcium communication between the spine head and the parent dendrite. We examined this hypothesis by local flash-photolysis of caged [Ca2+]i within dendrites of cultured hippocampal neurons. Cells were transfected with pDsred and were preincubated with caged EGTA AM (7uM) and Fluo-4 AM (2uM) for 1.5 hr at room temperature and imaged in a confocal microscope. Pulses of 4ns UV laser light focused in a spot of about 1um3 were aimed at a randomly selected segment of dendritic tree through a 63x water immersion objective. UV flashes produced [Ca2+]i transients in dendrites which peaked at 1-2 ms and decayed exponentially with fast (8-10 msec) and slow (30-40 msec) time constants. There was a slightly slower decay in thick (>2.5um) than in thin (<1.5um) dendrites. The slower component of decay could be eliminated by thapsigargin which blocks the calcium stores in the neuron. Diffusion of calcium in dendrites of all sizes and locations did not vary significantly, being symmetric and reaching about 3-3.5um on both sides of the uncaging spot. Interestingly, presence of a dendritic spine at the focus of uncaging reduced [Ca2+]i spread while the focal transient remained unaffected. Finally, [Ca2+]i diffused 4 times further from the uncaging spot (~12 um) in a thin glass tube of a similar diameter to that of a dendrite, indicating that both obstacles in the path and calcium uptake mechanisms in the dendrite retard the diffusion of calcium away from its origin.

*****

Yosef Shaul, Weizmann Institute of Science
On the dilemma of vectorial mobility versus diffusion in the nucleus

Nuclear mobility of macromolecules is by large metabolic energy-independent and believed to occur by a diffusion-based, passive, spatially random mechanism. We have evidence for an intranuclear vectorial mobility of a member of the p53 protein family towards one or both nuclear poles. Pharmacological disruptions of microtubules and actin filaments do not affect the mobility. The N- and C-terminal are essential for mobility and mutants lacking these regions, are neither polarized nor accumulate in any form. Remarkably, point mutants at the DNA-binding domain that are inactive in supporting transcription and apoptosis do not polarize but yet accumulate in confined structures of nuclear inclusions. These data suggest nuclear vectorial mobility of a functional protein towards the poles. The question is whether random diffusion may lead to polarized accumulation. In a second project we quantify the repair of DNA double strand breaks and the involvement of c-Abl tyrosine kinase in this process. Our hypothesis is that the free DNA ends are subjected to diffusion that may lead to ligation of two not linear ends, a process that causes genome rearrangement and instability. C-Abl kinase activity is increased with time after DNA breaks to reach a threshold of activity that stops the repair. The level of DNA rearrangement therefore is the function of c-Abl kinase activation rate and DNA diffusion.