The translational mobility of substances within the cytoplasmic matrix.

The translational mobility of fluorescent-labeled molecules injected into the cytoplasm of living cells can be measured by the fluorescence recovery after photobleaching (FRAP) technique. In the fibroblast cytoplasm, the diffusion coefficients, D, of test macromolecules ranging in molecular weight from 12,000 to 440,000 are about 10(-8) cm2/sec and exhibit almost no dependence on molecular weight. FRAP experiments also showed that macromolecular diffusion within Sepharose beads having an effective pore size smaller than the "microtrabecular lattice" is only slightly retarded compared to buffer values--in contrast to the marked retardation measured in the cytoplasm. This leads to the conclusion that diffusion in the cytomatrix is dominated not by steric effects but rather by binding of the diffusing species to elements of the cytomatrix. These diffusion rates were difficult to modulate; cytochalasin, colchicine (except at 5 degrees C), and taxol treatments had little effect. The diffusion rates were not dependent on cellular energy metabolism. However, hypotonic treatment increased the D for bovine serum albumin by nearly 2-fold, whereas hypertonic treatment halved D. Withdrawing the free water from the cell by using 44% polyethylene glycol treatment stopped the translational mobility of the test molecules. A survey of the recent literature is presented, which shows that major differences in the cytomatrix of different cell types exist with respect to the translational diffusion of injected probes. Finally, the spectrum of cytoplasmic translational mobilities ranging from small molecules to organelles is discussed

The translational mobility of substances within the cytoplasmic matrix.

The translational mobility of fluorescent-labeled molecules injected into the cytoplasm of living cells can be measured by the fluorescence recovery after photobleaching (FRAP) technique. In the fibroblast cytoplasm, the diffusion coefficients, D, of test macromolecules ranging in molecular weight from 12,000 to 440,000 are about 10(-8) cm2/sec and exhibit almost no dependence on molecular weight. FRAP experiments also showed that macromolecular diffusion within Sepharose beads having an effective pore size smaller than the "microtrabecular lattice" is only slightly retarded compared to buffer values--in contrast to the marked retardation measured in the cytoplasm. This leads to the conclusion that diffusion in the cytomatrix is dominated not by steric effects but rather by binding of the diffusing species to elements of the cytomatrix. These diffusion rates were difficult to modulate; cytochalasin, colchicine (except at 5 degrees C), and taxol treatments had little effect. The diffusion rates were not dependent on cellular energy metabolism. However, hypotonic treatment increased the D for bovine serum albumin by nearly 2-fold, whereas hypertonic treatment halved D. Withdrawing the free water from the cell by using 44% polyethylene glycol treatment stopped the translational mobility of the test molecules. A survey of the recent literature is presented, which shows that major differences in the cytomatrix of different cell types exist with respect to the translational diffusion of injected probes. Finally, the spectrum of cytoplasmic translational mobilities ranging from small molecules to organelles is discussed

Modeling Dual Pathways for the Metazoan Spindle Assembly Checkpoint

Using computational modelling, we investigate mechanisms of signal transduction focusing on the spindle assembly checkpoint where a single unattached kinetochore is able to signal to prevent cell cycle progression. This inhibitory signal switches off rapidly once spindle microtubules have attached to all kinetochores. This requirement tightly constrains the possible mechanisms. Here we investigate two possible mechanisms for spindle checkpoint operation in metazoan cells, both supported by recent experiments. The first involves the free diffusion and sequestration of cell-cycle regulators. This mechanism is severely constrained both by experimental fluorescence recovery data and also by the large volumes involved in open mitosis in metazoan cells. Using a simple mathematical analysis and computer simulation, we find that this mechanism can generate the inhibition found in experiment but likely requires a two stage signal amplification cascade. The second mechanism involves spatial gradients of a short-lived inhibitory signal that propagates first by diffusion but then primarily via active transport along spindle microtubules. We propose that both mechanisms may be operative in the metazoan spindle assembly checkpoint, with either able to trigger anaphase onset even without support from the other pathway.Comment: 9 pages, 2 figure

Diffusion of injected macromolecules within the cytoplasm of living cells.

The diffusion of macromolecules introduced into the cytoplasm of human fibroblasts by erythrocyte-mediated microinjection was measured by the fluorescence recovery after photobleaching technique. The apparent diffusion coefficients for fluorescein-labeled IgG and fluorescein-labeled bovine serum albumin were approximately 10(-8) cm2/sec at 22 degrees C, consistent with the kinetics of spreading of the fluorescent probe following microinjection and approximately 1/70 the values in aqueous buffer. The diffusion of labeled bovine serum albumin was shown to be strongly dependent on temperature and, in fact, similar to that expected in a 61% aqueous sucrose solution. However, the marked reduction in diffusion at 5 degrees C could be fully reversed by incubation with 0.1 mM colchicine. These findings suggest that cytoplasmic diffusion rates are reduced relative to rates in aqueous media as a result of increased aqueous phase viscosity or the impedence provided by structural elements. Several simple models to account for the data are presented

From the cell membrane to the nucleus: unearthing transport mechanisms for Dynein

Mutations in the motor protein cytoplasmic dynein have been found to cause Charcot-Marie-Tooth disease, spinal muscular atrophy, and severe intellectual disabilities in humans. In mouse models, neurodegeneration is observed. We sought to develop a novel model which could incorporate the effects of mutations on distance travelled and velocity. A mechanical model for the dynein mediated transport of endosomes is derived from first principles and solved numerically. The effects of variations in model parameter values are analysed to find those that have a significant impact on velocity and distance travelled. The model successfully describes the processivity of dynein and matches qualitatively the velocity profiles observed in experiments

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization

Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma

<p>Abstract</p> <p>Background</p> <p>The etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease.</p> <p>Methods</p> <p>We first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR.</p> <p>Results</p> <p>Each of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma).</p> <p>Conclusions</p> <p>The data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells.</p