Thermomechanical analysis of collagen crosslinking in the developing lamb pericardium

In a developing lamb model, we have used hydrothermal isometric tension (HIT) techniques to assess collagen crosslink stability and its contribution to the mechanical properties of the pericardium. Strip samples of tissue were either: (i) heated to a 90 degrees C isotherm or (ii) heated in 5 degrees C increments between 60-90 degrees C and then 93 and 97 degrees C isotherms. The half-life of stress relaxation associated with peptide bond hydrolysis (t1/2) was calculated at each isotherm. The activation energy, Eact, for the hydrolysis-associated relaxation process was also calculated using the data from the stepwise HIT tests--a technical improvement which significantly reduces the experimental time required to develop statistically valid measurements. Crosslinking in the pericardium increased during development and was demonstrated both by thermoelasticity and by resistance to enzymatic solubilization. We observed greater conversation to thermally stable crosslinks upon maturation, the ratio of the NaBH4-stabilized/unstabilized half-lives peaking at 21 days postnatal. Whereas tissue from lambs (119 day fetal, and 3 day and 21 day postpartum) showed an early maximum and rapid decay of force, NaBH4 stabilization significantly increased thermal stability and yielded profiles similar to those in adult tissue.     

Delineation of discrete population segments of shortnose sturgeon <Emphasis Type="Italic">Acipenser brevirostrum</Emphasis> based on mitochondrial DNA control region sequence analysis

Shortnose sturgeon Acipenser brevirostrum is federally listed as ‘‘an endangered species threatened with extinction’’ in the U.S. but its listing status is currently under review. As part of this process, the U.S. National Marine Fisheries Service will determine if shortnose sturgeon are divided into Distinct Population Segments (DPS) across its distribution. In this regard, we sought to determine if shortnose sturgeon occur in genetically “discrete population segments,” and if so, the boundaries of each. We used mitochondrial DNA (mtDNA) control region sequence analysis to assess the genetic discreteness of 14 of 19 river populations that were recommended as DPS in the 1998 Final Recovery Plan for Shortnose Sturgeon. Nine of the 14 proposed DPS proved significantly discrete (P < 0.05 after Bonferoni correction) from both of their bracketing populations, the exceptions being those in the Penobscot River, Chesapeake Bay, Cooper River, and Ogeechee River (our sample from the Cape Fear River was insufficient to statistically analyze). Haplotype frequencies in the newly “rediscovered” Penobscot River collection were almost identical to those in the proximal Kennebec River system. Genetic data in combination with tagging results suggest that shortnose sturgeon in the Penobscot River are probably migrants from the Kennebec. Likewise, shortnose sturgeon found today within the Chesapeake Bay appear to be migrants from the Delaware River. While haplotype frequencies in the remnant Santee River population in Lake Marion differed significantly from those in nearby Winyah Bay, they did not differ significantly from those in the Cooper River. This suggests that the Cooper River harbors descendants of the Santee River population that are unable to access their historical spawning locales. The Ogeechee River collection was not genetically distinct from that in the nearby Savannah River, suggesting that it may host descendants of hatchery-reared individuals of Savannah River ancestry. Our genetic results indicate that most, but not all, rivers with shortnose sturgeon host genetically discrete populations, constituting important information in the consideration of DPS designations. However, shortnose sturgeon migrations through coastal waters to proximal rivers and release of hatchery-reared fish may confound results from genetic studies such as ours and lead to the possible misidentification of discrete population segments.     

Linking Changes in Epithelial Morphogenesis to Cancer Mutations Using Computational Modeling

Most tumors arise from epithelial tissues, such as mammary glands and lobules, and their initiation is associated with the disruption of a finely defined epithelial architecture. Progression from intraductal to invasive tumors is related to genetic mutations that occur at a subcellular level but manifest themselves as functional and morphological changes at the cellular and tissue scales, respectively. Elevated proliferation and loss of epithelial polarization are the two most noticeable changes in cell phenotypes during this process. As a result, many three-dimensional cultures of tumorigenic clones show highly aberrant morphologies when compared to regular epithelial monolayers enclosing the hollow lumen (acini). In order to shed light on phenotypic changes associated with tumor cells, we applied the bio-mechanical IBCell model of normal epithelial morphogenesis quantitatively matched to data acquired from the non-tumorigenic human mammary cell line, MCF10A. We then used a high-throughput simulation study to reveal how modifications in model parameters influence changes in the simulated architecture. Three parameters have been considered in our study, which define cell sensitivity to proliferative, apoptotic and cell-ECM adhesive cues. By mapping experimental morphologies of four MCF10A-derived cell lines carrying different oncogenic mutations onto the model parameter space, we identified changes in cellular processes potentially underlying structural modifications of these mutants. As a case study, we focused on MCF10A cells expressing an oncogenic mutant HER2-YVMA to quantitatively assess changes in cell doubling time, cell apoptotic rate, and cell sensitivity to ECM accumulation when compared to the parental non-tumorigenic cell line. By mapping in vitro mutant morphologies onto in silico ones we have generated a means of linking the morphological and molecular scales via computational modeling. Thus, IBCell in combination with 3D acini cultures can form a computational/experimental platform for suggesting the relationship between the histopathology of neoplastic lesions and their underlying molecular defects.     

NHERFs, NEP, MAGUKs, and more: interactions that regulate PTEN

This year marks the 10th anniversary of the discovery of the PTEN/MMAC1/TEP1 tumor suppressor gene (hereafter referred to as PTEN), one of the most commonly mutated genes in cancer. PTEN encodes a lipid phosphatase that dephosphorylates phosphoinositide-3,4,5-triphosphate (PIP(3)), thereby counteracting mitogenic signaling pathways driven by phosphoinositol-3-kinases (PI3K). By opposing PI3K signaling, PTEN inhibits the activation of the critical PI3K effector proteins Akt1-3 (also known as protein kinase B or PKB). Given its central role in antagonizing PI3K signaling, one might expect that like PI3K, the activity of the PTEN protein would be highly regulated by numerous protein/protein interactions. However, surprisingly little is known about such interactions. This fact, combined with the generally accepted notion that phosphatases are less exquisitely regulated than kinases, has led to the idea that PTEN may function in a relatively unregulated fashion. Here we review the identities and proposed functions of known PTEN-interacting proteins, and point out avenues of investigation that we hope may be fruitful in identifying important new mechanisms of PTEN regulation in mammalian cells.     

BACE1 regulates voltage-gated sodium channels and neuronal activity

BACE1 activity is significantly increased in the brains of Alzheimer's disease patients, potentially contributing to neurodegeneration. The voltage-gated sodium channel (Na(v)1) beta2-subunit (beta2), a type I membrane protein that covalently binds to Na(v)1 alpha-subunits, is a substrate for BACE1 and gamma-secretase. Here, we find that BACE1-gamma-secretase cleavages release the intracellular domain of beta2, which increases mRNA and protein levels of the pore-forming Na(v)1.1 alpha-subunit in neuroblastoma cells. Similarly, endogenous beta2 processing and Na(v)1.1 protein levels are elevated in brains of BACE1-transgenic mice and Alzheimer's disease patients with high BACE1 levels. However, Na(v)1.1 is retained inside the cells and cell surface expression of the Na(v)1 alpha-subunits and sodium current densities are markedly reduced in both neuroblastoma cells and adult hippocampal neurons from BACE1-transgenic mice. BACE1, by cleaving beta2, thus regulates Na(v)1 alpha-subunit levels and controls cell-surface sodium current densities. BACE1 inhibitors may normalize membrane excitability in Alzheimer's disease patients with elevated BACE1 activity.     

Invadolysin acts genetically via the SAGA complex to modulate chromosome structure

Identification of components essential to chromosome structure and behaviour remains a vibrant area of study. We have previously shown that invadolysin is essential in Drosophila, with roles in cell division and cell migration. Mitotic chromosomes are hypercondensed in length, but display an aberrant fuzzy appearance. We additionally demonstrated that in human cells, invadolysin is localized on the surface of lipid droplets, organelles that store not only triglycerides and sterols but also free histones H2A, H2Av and H2B. Is there a link between the storage of histones in lipid droplets and the aberrantly structured chromosomes of invadolysin mutants? We have identified a genetic interaction between invadolysin and nonstop, the de-ubiquitinating protease component of the SAGA (Spt-Ada-Gcn5-acetyltransferase) chromatin-remodelling complex. invadolysin and nonstop mutants exhibit phenotypic similarities in terms of chromosome structure in both diploid and polyploid cells. Furthermore, IX-14¹/not¹ transheterozygous animals accumulate mono-ubiquitinated histone H2B (ubH2B) and histone H3 tri-methylated at lysine 4 (H3K4me3). Whole mount immunostaining of IX-14¹/not¹ transheterozygous salivary glands revealed that ubH2B accumulates surprisingly in the cytoplasm, rather than the nucleus. Over-expression of the Bre1 ubiquitin ligase phenocopies the effects of mutating either the invadolysin or nonstop genes. Intriguingly, nonstop and mutants of other SAGA subunits (gcn5, ada2b and sgf11) all suppress an invadolysin-induced rough eye phenotype. We conclude that the abnormal chromosome phenotype of invadolysin mutants is likely the result of disrupting the histone modification cycle, as accumulation of ubH2B and H3K4me3 is observed. We further suggest that the mislocalization of ubH2B to the cytoplasm has additional consequences on downstream components essential for chromosome behaviour. We therefore propose that invadolysin plays a crucial role in chromosome organization via its interaction with the SAGA complex.     

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.