Pure F-actin networks are distorted and branched by steps in the critical-point drying method

Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility.     

Glycogen metabolism in tissues from a mouse model of Lafora disease

Laforin, encoded by the EPM2A gene, by sequence is a member of the dual specificity protein phosphatase family. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons, muscle and other tissues. Glycogen metabolizing enzymes were analyzed in a transgenic mouse over-expressing a dominant negative form of laforin that accumulates Lafora bodies in several tissues. Skeletal muscle glycogen was increased 2-fold as was the total glycogen synthase protein. However, the -/+glucose-6-P activity of glycogen synthase was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Although there were significant differences in enzyme activities in muscle, the results do not support the hypothesis that Lafora body formation is caused by a major change in the balance between glycogen elongation and branching activities.     

PTEN modulates GDNF/RET mediated chemotaxis and branching morphogenesis in the developing kidney

The RET receptor tyrosine kinase is activated by GDNF and controls outgrowth and invasion of the ureteric bud epithelia in the developing kidney. In renal epithelial cells and in enteric neuronal precursor cells, activation of RET results in chemotaxis as Ret expressing cells invade the surrounding GDNF expressing tissue. One potential downstream signaling pathway governing RET mediated chemotaxis may require phosphatidylinositol 3-kinase (PI3K), which generates PI(3,4,5) triphosphate. The PTEN tumor suppressor gene encodes a protein and lipid phosphatase that regulates cell growth, apoptosis and many other cellular processes. PTEN helps regulate cellular chemotaxis by antagonizing the PI3K signaling pathway through dephosphorylation of phosphotidylinositol triphosphates. In this report, we show that PTEN suppresses RET mediated cell migration and chemotaxis in cell culture assays, that RET activation results in asymmetric localization of inositol triphosphates and that loss of PTEN affects the pattern of branching morphogenesis in developing mouse kidneys. These data suggest a critical role for the PI3K/PTEN axis in shaping the pattern of epithelial branches in response to RET activation.     

Morphogenesis of epithelial tubes: Insights into tube formation, elongation, and elaboration

Epithelial tubes are a fundamental tissue across the metazoan phyla and provide an essential functional component of many of the major organs. Recent work in flies and mammals has begun to elucidate the cellular mechanisms driving the formation, elongation, and branching morphogenesis of epithelial tubes during development. Both forward and reverse genetic techniques have begun to identify critical molecular regulators for these processes and have revealed the conserved role of key pathways in regulating the growth and elaboration of tubular networks. In this review, we discuss the developmental programs driving the formation of branched epithelial networks, with specific emphasis on the trachea and salivary gland of Drosophila melanogaster and the mammalian lung, mammary gland, kidney, and salivary gland. We both highlight similarities in the development of these organs and attempt to identify tissue and organism specific strategies. Finally, we briefly consider how our understanding of the regulation of proliferation, apicobasal polarity, and epithelial motility during branching morphogenesis can be applied to understand the pathologic dysregulation of these same processes during metastatic cancer progression.     

Stochasticity, invasions, and branching random walks

We link deterministic integrodifference equations to stochastic, individual-based simulations by means of branching random walks. Using standard methods, we determine speeds of invasion for both average densities and furthest-forward individuals. For density-independent branching random walks, demographic stochasticity can produce extinction. Demographic stochasticity does not, however, reduce the overall asymptotic speed of invasion or preclude continually accelerating invasions.     

Role for laminin-alpha5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4, perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the laminin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of laminin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4.     

Statistical and Frequency-Amplitude Characteristics of Ultraweak Emissions of the Loach Eggs and Embryos under the Normal Conditions and during Their Optic Interactions. 1. Characteristics of Ultraweak Emission in Normal Development and the Optic Role of Egg Envelopes

Ultraweak emissions of groups comprising several dozens of unfertilized and fertilized loach eggs, embryos, larvae, and their egg envelopes were measured on a photomultiplier tube. The envelopes absorbed the light from external sources but readily gave it back in the absence of embryos. We carried out statistical and frequency-amplitude analyses of ultraweak emissions and studied the autocorrelation structure of their frequency spectra. The frequencies of signals with different intensity underwent regular changes during development. Cascades of short-term (1 ms) flashes timed (during cleavage) to furrowing were a characteristic element of ultraweak emission. The Fourier spectra of developing embryos had pronounced frequency-amplitude peaks and higher, than in unfertilized eggs and inanimate samples, mutual correlation during successive time intervals. Stage-specific translational symmetry of the frequency spectra of ultraweak emissions was demonstrated, which suggests the presence in groups of embryos of a coordinated system of harmonic oscillators. The latter underwent regular changes during development. The measurement of ultraweak emissions represents a unique non-invasive method of analysis of these oscillators.     

The distribution of surviving blocks of an ancestral genome

What is the chance that some part of a stretch of genome will survive? In a population of constant size, and with no selection, the probability of survival of some part of a stretch of map length y < 1 approaches y/log(yt/2) for log(yt) > or = 1. Thus, the whole genome is certain to be lost, but the rate of loss is extremely slow. This solution extends to give the whole distribution of surviving block sizes as a function of time. We show that the expected number of blocks at time t is 1+yt and give expressions for the moments of the number of blocks and the total amount of genome that survives for a given time. The solution is based on a branching process and assumes complete interference between crossovers, so that each descendant carries only a single block of ancestral material. We consider cases where most individuals carry multiple blocks, either because there are multiple crossovers in a long genetic map, or because enough time has passed that most individuals in the population are related to each other. For species such as ours, which have a long genetic map, the genome of any individual which leaves descendants (approximately 80% of the population for a Poisson offspring number with mean two) is likely to persist for an extremely long time, in the form of a few short blocks of genome.     

Establishment success and extinction risk in autocorrelated environments

We consider establishment success (and extinction risk of small populations) in fluctuating environments, by means of an inhomogeneous branching process model. In this model it is assumed that individuals reproduce asexually during discrete reproduction periods. Within each period individuals reproduce independently and have random numbers of offspring. Expected numbers of offspring vary over reproduction periods due to random environmental changes. Previous simulation results indicated that there is a positive autocorrelation between the establishment probabilities of invaders in successive reproduction periods when environmental states are independently distributed. This result was never formally proved. In this paper we prove that this is indeed true, regardless of the form of the distribution of environmental states or the offspring distribution (under a monotonicity condition, which holds for biologically realistic models). Furthermore, we prove that it is also true for positively autocorrelated environmental states. We show by a counterexample that in environments with a strong negative autocorrelation establishment probabilities can be negatively autocorrelated. This was further examined through simulations. Our results imply that in independent, positively autocorrelated and weakly negatively autocorrelated environments the probability of success of invasion in different independently varying sites is the highest, followed by sequential invasion. For environments with a strong negative autocorrelation, sequential invasion has the highest probability of success. Effects of autocorrelation were further examined with simulations. From the results it appears that the expected length of 'runs of bad luck' is the most crucial factor for establishment success.     

Cadmium,nickel, copper,and zinc influence on microfilament organization in Arabidopsis root cells

The plant cytoskeleton orchestrates such fundamental processes in cells as division, growth and development, polymer cross-linking, membrane anchorage, etc. Here, we describe the influence of Cd²⁺, Ni²⁺, Zn²⁺, and Cu²⁺ on root development and vital organization of actin filaments into different cells of Arabidopsis thaliana line expressing GFP-FABD2. CdSO₄, NiSO₄, CuSO₄, and ZnSO₄ were used in concentrations of 5–20 µM in this study. It was found that Cd, Ni, and Cu cause dose-dependent primary root growth inhibition and alteration of the root morphology, whereas Zn slightly stimulates root growth and does not affect the morphology of Arabidopsis roots. This growth inhibition/stimulation correlated with the various sensitivities of microfilaments to Cd, Ni, Cu, and Zn action. It was established that Cd, Ni, and Cu affected predominantly the actin filaments of meristematic cells. Cells of transition and elongation zones demonstrated strong actin filament sensitivity to Cd and Cu. Microfilaments of elongating root cells were more sensitive to Ni and Cu. Although Cd, Ni, and Cu stimulated root hair growth after long-term treatment, actin filaments were destroyed after 1 h exposure with these metals. Zn did not disrupt native actin filament organization in root cells. Thus, our investigation shows that microfilaments act as sensitive cellular targets for Cd, Ni, and Cu. More data on effects on native actin filaments organization would contribute to a better understanding of plant tolerance mechanisms to the action of these metals.