Interactions of tropomyosin Tpm1.1 on a single actin filament: A method for extraction and processing of high resolution TIRF microscopy data.

Skeletal muscle tropomyosin (Tpm1.1) is an elongated, rod-shaped, alpha-helical coiled-coil protein that forms continuous head-to-tail polymers along both sides of the actin filament. In this study we use single molecule fluorescence TIRF microscopy combined with a microfluidic device and fluorescently labelled proteins to measure Tpm1.1 association to and dissociation from single actin filaments. Our experimental setup allows us to clearly resolve Tpm1.1 interactions on both sides of the filaments. Here we provide a semi-automated method for the extraction and quantification of kymograph data for individual actin filaments bound at different Tpm1.1 concentrations. We determine boundaries on the kymograph on each side of the actin filament, based on intensity thresholding, performing fine manual editing of the boundaries (if needed) and extracting user defined kinetic properties of the system. Using our analytical tools we can determine (i) nucleation point(s) and rates, (ii) elongation rates of Tpm1.1, (iii) identify meeting points after the saturation of filament, and when dissociation occurs, (iv) initiation point(s), (v) the final dissociation point(s), as well as (vi) dissociation rates. All of these measurements can be extracted from both sides of the filament, allowing for the determination of possible differences in behaviour on the two sides of the filament, and across concentrations. The robust and repeatable nature of the method allows quantitative, semi-automated analyses to be made of large studies of acto-tropomyosin interactions, as well as for other actin binding proteins or filamentous structures, opening the way for dissection of the dynamics underlying these interactions.

CASZ1 inhibits cell cycle progression in neuroblastoma by restoring pRb activity.

Dysregulation of cell cycle genes such as Cyclin D1 and Chk1 contributes to the undifferentiated phenotype of neuroblastoma (NB). CASZ1 functions as a tumor suppressor in NB; here we sought to determine how loss of CASZ1 contributes to cell cycle dysregulation in NB. CASZ1 restoration in NB cells delays NB cell cycle progression. The earliest changes occur within 8 h of CASZ1 restoration in SY5Y cells with a 2.8-fold increase in the level of p21, an inhibitor of Cdk2/4. By 16 h, there is a 40% decrease in the steady-state levels of Cdk6. Restoration of CASZ1 decreases Cdk2-dependent cyclins A and E protein levels and Cdk4/6-dependent Cyclin D1 protein levels. The restoration of CASZ1 resulted in a decrease in pRb phosphorylation and a significant reduction of E2F transcriptional activity. Subsequent to the changes in the G 1/S transition, induction of CASZ1 results in a decrease in Cyclin B levels and Cdc25c phosphatase levels, an upstream activator of the G 2/M regulator CyclinB:Cdk1. In addition, induction of CASZ1 results in a decrease in the levels of phospho-Chk1, a key M-phase regulatory kinase. Similar results were found in a NB cell line with MYCN amplification. Taken together, this study indicates that restoration of CASZ1 activates pRb in G 1 and inhibits the G 2/M regulators Cyclin B1 and Chk1, leading to a lengthening of NB cell cycle progression and a subsequent decrease in cell proliferation.

Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation.

This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM.

MYCN and the epigenome.

It is well known that Neuroblastoma (NB) patients whose tumors have an undifferentiated histology and a transcriptome enriched in cell cycle genes have a worse prognosis. This contrasts with the good prognoses of patients whose tumors have histologic evidence of differentiation and a transcriptome enriched in differentiation genes. Tumor cell lines from poor prognosis, high-risk patients contain a number of genetic alterations, including amplification of MYCN, 1pLOH, and unbalanced 11q or gains of Chr 17 and 7, and exhibit uncontrolled growth and an undifferentiated phenotype in in vitro culture. Yet treatment of such NB cell lines with retinoic acid results in growth control and induction of differentiation. This indicates that the signaling pathways that regulate cell growth and differentiation are not functionally lost but dysregulated. Agents such as retinoic acid normalize the signaling pathways and impose growth control and induction of differentiation. Recent studies in embryonic stem cells indicate that polycomb repressor complex proteins (PRC1 and PRC2) play a major role in regulating stem cell lineage specification and coordinating the shift from a transcriptome that supports self-renewal or growth to one that specifies lineage and controls growth. We have shown that in NB, the PRC2 complex is elevated in undifferentiated NB tumors and functions to suppress a number of tumor suppressor genes. This study will review the role of MYC genes in regulating the epigenome in normal development and explore how this role may be altered during tumorigenesis.

Transgenic mitochondrial superoxide dismutase and mitochondrially targeted catalase prevent antiretroviral-induced oxidative stress and cardiomyopathy.

Transgenic mice (TG) were used to define mitochondrial oxidative stress and cardiomyopathy (CM) induced by zidovudine (AZT), an antiretroviral used to treat HIV/AIDS. Genetically engineered mice either depleted or overexpressed mitochondrial superoxide dismutase (SOD2(+/-) KOs and SOD2-OX, respectively) or expressed mitochondrially targeted catalase (mCAT). TGs and wild-type (WT) littermates were treated (oral AZT, 35 days). Cardiac mitochondrial H(2)O(2), aconitase activity, histology and ultrastructure were analyzed. Left ventricle (LV) mass and LV end-diastolic dimension were determined echocardiographically. AZT induced cardiac oxidative stress and LV dysfunction in WTs. Cardiac mitochondrial H(2)O(2) increased and aconitase was inactivated in SOD2(+/-) KOs, and cardiac dysfunction was worsened by AZT. Conversely, the cardiac function in SOD2-OX and mCAT hearts was protected. In SOD2-OX and mCAT TG hearts, mitochondrial H(2)O(2), LV mass and LV cavity volume resembled corresponding values from vehicle-treated WTs. AZT worsens cardiac dysfunction and increases mitochondrial H(2)O(2) in SOD2(+/-) KO. Conversely, both SOD2-OX and mCAT TGs prevent or attenuate AZT-induced cardiac oxidative stress and LV dysfunction. As dysfunctional changes are ameliorated by decreasing and worsened by increasing H(2)O(2) abundance, oxidative stress from H(2)O(2) is crucial pathogenetically in AZT-induced mitochondrial CM.