Pseudopod growth and evolution during cell movement is controlled through SCAR/WAVE dephosphorylation

Regulated dephosphorylation of a fraction of the cellular SCAR pool is a key step in SCAR activation during pseudopod growth. Phosphorylation increases autoinhibition of the intact complex. Dephosphorylation weakens this interaction and facilitates SCAR activation but also destabilizes the protein. We show that SCAR is specifically dephosphorylated in pseudopods, increasing activation by Rac and lipids and supporting positive feedback of pseudopod growth.

Loss of Dictyostelium HSPC300 causes a scar-like phenotype and loss of SCAR protein.

HSPC300 is essential for most SCAR complex functions. The phenotype of HSPC300 knockouts is most similar to mutants in scar, not the other members of the SCAR complex, suggesting that HSPC300 acts most directly on SCAR itself.

Abi mutants in Dictyostelium reveal specific roles for the SCAR/WAVE complex in cytokinesis.

Actin polymerization drives multiple cell processes involving movement and shape change. SCAR/WAVE proteins connect signaling to actin polymerization through the activation of the Arp2/3 complex. SCAR/WAVE is normally found in a complex with four other proteins: PIR121, Nap1, Abi2,and HSPC300 (Figure S1A available online) [1-3]. However,there is no consensus as to whether the complex functions as an unchanging unit or if it alters its composition in response to stimulation, as originally proposed by Edenet al. [1]. It also is unclear whether complex members exclusively regulate SCAR/WAVEs or if they have additional targets [4-6]. Here, we analyze the roles of the unique Dictyostelium Abi. We find that abiA null mutants show less severe defects in motility than do scar null cells, indicating--unexpectedly--that SCAR retains partial activity in the absence of Abi. Furthermore, abiA null mutants have a serious defect in cytokinesis, which is not seen in other SCAR complex mutants and is seen only when SCAR itself is present. Detailed examination reveals that normal cytokinesis requires SCAR activity, apparently regulated through multiple pathways.

Cell motility and SCAR localisation in axenically growing Dictyostelium cells.

Dictyostelium is a popular experimental organism, in particular for studies of actin dynamics, cell motility and chemotaxis. We find that the motility of axenic cells is unexpectedly different from other strains during growth. In particular, vegetative AX3 cells do not show detectable localisation of SCAR and its regulatory complex to actin-rich protrusions such as filopodia and pseudopodia. Similarly, a range of different mutations, in particular knockouts of members of the SCAR complex and Ras proteins, cause different phenotypes during vegetative growth in different parental strains. Development reverses this unusual behaviour; aggregation-competent AX3 cells localise SCAR in the same way as cells of other strains and species. Studies on cell motility using vegetative cells should therefore be interpreted with caution.

Regulation of actin assembly by SCAR/WAVE proteins.

Actin reorganization is a tightly regulated process that co-ordinates complex cellular events, such as cell migration, chemotaxis, phagocytosis and adhesion, but the molecular mechanisms that underlie these processes are not well understood. SCAR (suppressor of cAMP receptor)/WAVE [WASP (Wiskott-Aldrich syndrome protein)-family verprolin homology protein] proteins are members of the conserved WASP family of cytoskeletal regulators, which play a critical role in actin dynamics by triggering Arp2/3 (actin-related protein 2/3)-dependent actin nucleation. SCAR/WAVEs are thought to be regulated by a pentameric complex which also contains Abi (Abl-interactor), Nap (Nck-associated protein), PIR121 (p53-inducible mRNA 121) and HSPC300 (haematopoietic stem progenitor cell 300), but the structural organization of the complex and the contribution of its individual components to the regulation of SCAR/WAVE function remain unclear. Additional features of SCAR/WAVE regulation are highlighted by the discovery of other interactors and distinct complexes. It is likely that the combinatorial assembly of different components of SCAR/WAVE complexes will prove to be vital for their roles at the centre of dynamic actin reorganization.

Soils of contrasting pH affect the decomposition of buried mammalian (Ovis aries) skeletal muscle tissue

Little is known about the effect of edaphic conditions on the decomposition of buried mammalian tissues. To address this, we set up a replicated incubation study with three fresh soils of contrasting pH: a Podsol (acidic), a Cambisol (neutral), and a Rendzina (alkaline), in which skeletal muscle tissue (SMT) of known mass was allowed to decompose. Our results clearly demonstrated that soil type had a considerable effect on the decomposition of SMT buried in soil. Differences in the rate of decomposition were up to three times greater in the Podsol compared with the Rendzina. The rate of microbial respiration was correlated to the rate of soft tissue loss, which suggests that the decomposition of SMT is dependent on the microbial community present in the soil. Decompositional by-products caused the pH of the immediate soil environment to change, becoming more alkaline at first, before acidifying. Our results demonstrate the need for greater consideration of soil type in future taphonomic studies.

Does repeated burial of skeletal muscle tissue (Ovis aries) in soil affect subsequent decomposition?

The repeated introduction of an organic resource to soil can result in its enhanced degradation. This phenomenon is of primary importance in agroecosystems, where the dynamics of repeated nutrient, pesticide, and herbicide amendment must be understood to achieve optimal yield. Although not yet investigated, the repeated introduction of cadaveric material is an important area of research in forensic science and cemetery planning. It is not currently understood what effects the repeated burial of cadaveric material has on cadaver decomposition or soil processes such as carbon mineralization. To address this gap in knowledge, we conducted a laboratory experiment using ovine (Ovis aries) skeletal muscle tissue (striated muscle used for locomotion) and three contrasting soils (brown earth, rendzina, podsol) from Great Britain. This experiment comprised two stages. In Stage I skeletal muscle tissue (150 g as 1.5 g cubes) was buried in sieved (4.6 mm) soil (10 kg dry weight) calibrated to 60% water holding capacity and allowed to decompose in the dark for 70 days at 22 °C. Control samples comprised soil without skeletal muscle tissue. In Stage II, soils were weighed (100 g dry weight at 60% WHC) into 1285 ml incubation microcosms. Half of the soils were designated for a second tissue amendment, which comprised the burial (2.5 cm) of 1.5 g cube of skeletal muscle tissue. The remaining half of the samples did not receive tissue. Thus, four treatments were used in each soil, reflecting all possible combinations of tissue burial (+) and control (−). Subsequent measures of tissue mass loss, carbon dioxide-carbon evolution, soil microbial biomass carbon, metabolic quotient and soil pH show that repeated burial of skeletal muscle tissue was associated with a significantly greater rate of decomposition in all soils. However, soil microbial biomass following repeated burial was either not significantly different (brown earth, podsol) or significantly less (rendzina) than new gravesoil. Based on these results, we conclude that enhanced decomposition of skeletal muscle tissue was most likely due to the proliferation of zymogenous soil microbes able to better use cadaveric material re-introduced to the soil.

Microbial decomposition of skeletal muscle tissue (Ovis aries) in a sandy loam soil at different temperatures

A laboratory experiment was conducted to determine the effect of temperature (2, 12, 22 °C) on the rate of aerobic decomposition of skeletal muscle tissue (Ovis aries) in a sandy loam soil incubated for a period of 42 days. Measurements of decomposition processes included skeletal muscle tissue mass loss, carbon dioxide (CO2) evolution, microbial biomass, soil pH, skeletal muscle tissue carbon (C) and nitrogen (N) content and the calculation of metabolic quotient (qCO2). Incubation temperature and skeletal muscle tissue quality had a significant effect on all of the measured process rates with 2 °C usually much lower than 12 and 22 °C. Cumulative CO2 evolution at 2, 12 and 22 °C equaled 252, 619 and 905 mg CO2, respectively. A significant correlation (P<0.001) was detected between cumulative CO2 evolution and tissue mass loss at all temperatures. Q10s for mass loss and CO2 evolution, which ranged from 1.19 to 3.95, were higher for the lower temperature range (Q10(2– 12 °C)>Q10(12–22 °C)) in the Ovis samples and lower for the low temperature range (Q10(2–12 °C)tissue mass loss and cumulative CO2 evolution suggest that tissue decomposition was most efficient at 2 °C. These phenomena may be due to lower microbial catabolic requirements at lower temperature.

A laboratory incubation method for determining the rate of microbiological degradation of skeletal muscle tissue in soil

A controlled laboratory experiment is described, in principle and practice, which can be used for the of determination the rate of tissue decomposition in soil. By way of example, an experiment was conducted to determine the effect of temperature (12°C, 22°C) on the aerobic decomposition of skeletal muscle tissue (Organic Texel × Suffolk lamb (Ovis aries)) in a sandy loam soil. Measurements of decomposition processes included muscle tissue mass loss, microbial CO2 respiration, and muscle tissue carbon (C) and nitrogen (N). Muscle tissue mass loss at 22°C always was greater than at 12°C (p < 0.001). Microbial respiration was greater in samples incubated at 22°C for the initial 21 days of burial (p < 0.01). All buried muscle tissue samples demonstrated changes in C and N content at the end of the experiment. A significant correlation (p < 0.001) was demonstrated between the loss of muscle tissue-derived C (C1) and microbially-respired C (Cm) demonstrating CO2 respiration may be used to predict mass loss and hence biodegradation. In this experiment Q10 (12°C - 22°C) = 2.0. This method is recommended as a useful tool in determining the effect of environmental variables on the rate of decomposition of various tissues and associated materials.

Bio-fabrication and physiological self-release of tissue equivalents using smart peptide amphiphile templates

In this study we applied a smart biomaterial formed from a self-assembling, multi-functional synthetic peptide amphiphile (PA) to coat substrates with various surface chemistries. The combination of PA coating and alignment-inducing functionalised substrates provided a template to instruct human corneal stromal fibroblasts to adhere, become aligned and then bio-fabricate a highlyordered, multi-layered, three-dimensional tissue by depositing an aligned, native-like extracellular matrix. The newly-formed corneal tissue equivalent was subsequently able to eliminate the adhesive properties of the template and govern its own complete release via the action of endogenous proteases. Tissues recovered through this method were structurally stable, easily handled, and carrier-free. Furthermore, topographical and mechanical analysis by atomic force microscopy showed that tissue equivalents formed on the alignment-inducing PA template had highly-ordered, compact collagen deposition, with a two-fold higher elastic modulus compared to the less compact tissues produced on the non-alignment template, the PA-coated glass. We suggest that this technology represents a new paradigm in tissue engineering and regenerative medicine, whereby all processes for the biofabrication and subsequent self-release of natural, bioprosthetic human tissues depend solely on simple templatetissue feedback interactions.

Phenylephrine and endothelin-1 upregulate connective tissue growth factor in neonatal rat cardiac myocytes.

Cardiac hypertrophy is associated with hypertrophic growth of cardiac myocytes and increased fibrosis. Much is known of the stimuli which promote myocyte hypertrophy and the changes associated with the response, but the links between the two are largely unknown. Using subtractive hybridization, we identified three genes which are acutely (<1 h) upregulated in neonatal rat ventricular myocytes exposed to the alpha-adrenergic agonist, phenylephrine. One represented connective tissue growth factor (CTGF) which is implicated in fibrosis and promotes hypertrophy in other cells. We further examined the expression of CTGF mRNA and protein in cardiac myocytes using quantitative PCR and immunoblotting, confirming that phenylephrine increased CTGF mRNA (maximal within 1 h) and protein (increased over 4 - 24 h). Endothelin-1 promoted a greater, though transient, increase in CTGF mRNA, but the increase in CTGF protein was sustained over 8 h. Neither agonist increased CTGF mRNA in cardiac non-myocytes. By increasing the expression of CTGF in cardiac myocytes, hypertrophic agonists such as phenylephrine and endothelin-1 may promote fibrosis. CTGF may also propagate the hypertrophic response initiated by these agonists.

Distribution analysis of the putative cancer marker S100A4 across invasive squamous cell carcinoma penile tissue

MS-based proteomic methods were utilised for the first time in the discovery of novel penile cancer biomarkers. MALDI MS imaging was used to obtain the in situ biomolecular MS profile of squamous cell carcinoma of the penis which was then compared to benign epithelial MS profiles. Spectra from cancerous and benign tissue areas were examined to identify MS peaks that best distinguished normal epithelial cells from invasive squamous epithelial cells, providing crucial evidence to suggest S100A4 to be differentially expressed. Verification by immunohistochemistry resulted in positive staining for S100A4 in a sub-population of invasive but not benign epithelial cells.