Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread. INTRODUCTIONMost plant viruses encode one or more proteins that are required to achieve local and systemic invasion of the host. These so-called movement proteins (MPs) enable viruses to exploit plasmodesmata, the gated, plasma membrane-lined channels that provide symplastic continuity between adjacent cells and through which plant cells communicate (Epel, 1994;Lucas and Gilbertson, 1994; Fenczik et al., 1995).Pioneering studies of MP functions were performed with the MP of tobacco mosaic virus (TMV) (Deom et al., 1987;Meshi et al., 1987). In infected tobacco plants as well as in transgenic plants, the MP accumulates in plasmodesmata and increases their size exclusion limit (Tomenius et al., 1987;Wolf et al., 1989; Atkins et al., 1991a; Ding et al., 1992;Moore et al., 1992;Oparka et al., 1997). The protein also binds single-stranded nucleic acids in vitro, resulting in unfolded and elongated protein-nucleic acid complexes.This observation led to the hypothesis that the virus moves from cell to cell in the form of a viral ribonucleoprotein complex (vRNP) that in size and structure is compatible with the modified plasmodesmata (Citovsky et al., 1990(Citovsky et al., , 1992.Although it is evident that the MP and vRNP must enlist cytoplasmic structures to aid transfer from their site of synthesis to the plasmodesmata, little is known about the nature of these components and about the targeting mechanism per se. F-actin and microtubules were proposed as targeting systems for MP (Heinlein et al., 1995;McLean et al., 1995; Carrington et al....

We present a 3D multi-cell simulation of a generic simplification of vascular tumor growth which can be easily extended and adapted to describe more specific vascular tumor types and host tissues. Initially, tumor cells proliferate as they take up the oxygen which the pre-existing vasculature supplies. The tumor grows exponentially. When the oxygen level drops below a threshold, the tumor cells become hypoxic and start secreting pro-angiogenic factors. At this stage, the tumor reaches a maximum diameter characteristic of an avascular tumor spheroid. The endothelial cells in the pre-existing vasculature respond to the pro-angiogenic factors both by chemotaxing towards higher concentrations of pro-angiogenic factors and by forming new blood vessels via angiogenesis. The tumor-induced vasculature increases the growth rate of the resulting vascularized solid tumor compared to an avascular tumor, allowing the tumor to grow beyond the spheroid in these linear-growth phases. First, in the linear-spherical phase of growth, the tumor remains spherical while its volume increases. Second, in the linear-cylindrical phase of growth the tumor elongates into a cylinder. Finally, in the linear-sheet phase of growth, tumor growth accelerates as the tumor changes from cylindrical to paddle-shaped. Substantial periods during which the tumor grows slowly or not at all separate the exponential from the linear-spherical and the linear-spherical from the linear-cylindrical growth phases. In contrast to other simulations in which avascular tumors remain spherical, our simulated avascular tumors form cylinders following the blood vessels, leading to a different distribution of hypoxic cells within the tumor. Our simulations cover time periods which are long enough to produce a range of biologically reasonable complex morphologies, allowing us to study how tumor-induced angiogenesis affects the growth rate, size and morphology of simulated tumors.

Somitogenesis, the formation of the body's primary segmental structure common to all vertebrate development, requires coordination between biological mechanisms at several scales. Explaining how these mechanisms interact across scales and how events are coordinated in space and time is necessary for a complete understanding of somitogenesis and its evolutionary flexibility. So far, mechanisms of somitogenesis have been studied independently. To test the consistency, integrability and combined explanatory power of current prevailing hypotheses, we built an integrated clock-and-wavefront model including submodels of the intracellular segmentation clock, intercellular segmentation-clock coupling via Delta/Notch signaling, an FGF8 determination front, delayed differentiation, clock-wavefront readout, and differential-cell-cell-adhesion-driven cell sorting. We identify inconsistencies between existing submodels and gaps in the current understanding of somitogenesis mechanisms, and propose novel submodels and extensions of existing submodels where necessary. For reasonable initial conditions, 2D simulations of our model robustly generate spatially and temporally regular somites, realistic dynamic morphologies and spontaneous emergence of anterior-traveling stripes of Lfng. We show that these traveling stripes are pseudo-waves rather than true propagating waves. Our model is flexible enough to generate interspecies-like variation in somite size in response to changes in the PSM growth rate and segmentation-clock period, and in the number and width of Lfng stripes in response to changes in the PSM growth rate, segmentation-clock period and PSM length.

A unique subfamily of calmodulin-dependent Ca 2؉ -ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca 2؉ -ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca 2؉ -ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg 2؉ , ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca 2؉ -ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals. Ca2ϩ is thought to function as an important second messenger in all eukaryotes (Bootman and Berridge, 1995;Clapham, 1995). In addition, Ca 2ϩ is required for the stability and activity of many proteins and appears to play a critical role in protein processing in the secretory pathway (Rudolph et al., 1989; Gill et al., 1996) Type IIA and IIB pumps include the "ER-type" and the "PM-type" Ca 2ϩ pumps, respectively, first characterized in animal cells. Previously, homologs of ER-or PM-type pumps were distinguished by three criteria: (a) localization to either the ER or PM, respectively, (b) differential sensitivity to inhibitors (e.g. ER-type inhibition by cyclopiazonic acid and thapsigargin), and (c) direct activation of PM-type pumps by calmodulin. However, not all plant homologs conform to these criteria (Bush, 1995;Evans and Williams, 1998).In plants several genes encoding type IIA pumps (ERtype homologs) have been cloned, including LCA1 from tomato (Wimmers et al., 1992), OsCA from rice (Chen et al., 1997), and ACA3/ECA1 (Arabidopsis Ca 2ϩ -ATPase, isoform 3/ER-Ca 2ϩ -ATPase isoform 1) from Arabidopsis (Liang et al., 1997). Consistent with the criteria for a typical ER-type pump, ACA3p (ACA isoform 3 protein) appears to reside in the ER (Liang et al., 1997). However, non-ER locations have been suggested for other isoforms. For example, Ferrol and Bennett (1996) obtained evidence for tonoplast and PM isoforms from membrane fractionation and immunodetection of pumps cross-reacting with an anti-LCA1 antibody.Three plant genes encoding type IIB pumps (PM...

We study the interface morphology of a 2D simulation of an avascular tumor composed of identical cells growing in an homogeneous healthy tissue matrix (TM), in order to understand the origin of the morphological changes often observed during real tumor growth. We use the GlazierGranerHogeweg model, which treats tumor cells as extended, deformable objects, to study the effects of two parameters: a dimensionless diffusion-limitation parameter defined as the ratio of the tumor consumption rate to the substrate transport rate, and the tumor-TM surface tension. We model TM as a nondiffusing field, neglecting the TM pressure and haptotactic repulsion acting on a real growing tumor; thus our model is appropriate for studying tumors with highly motile cells, e.g., gliomas. We show that the diffusion-limitation parameter determines whether the growing tumor develops a smooth (noninvasive) or fingered (invasive) interface, and that the sensitivity of tumor morphology to tumor-TM surface tension increases with the size of the dimensionless diffusion-limitation parameter. For large diffusion-limitation parameters we find a transition (missed in previous work) between dendritic structures, produced when tumor-TM surface tension is high, and seaweed-like structures, produced when tumor-TM surface tension is low. This observation leads to a direct analogy between the mathematics and dynamics of tumors and those observed in nonbiological directional solidification. Our results are also consistent with biological observation that hypoxia promotes invasive growth of tumor cells by inducing higher levels of receptors for scatter factors that weaken cell-cell adhesion and increase cell motility. These findings suggest that tumor