BMC Cell Biology

Apoptosis is a form of programmed cell death that is regulated by the Bcl-2 family and caspase family of proteins. The caspase cascade responsible for executing cell death following cytochromecrelease is well described; however the distinct roles of caspases-9, -3 and -7 during this process are not completely defined.

Here we demonstrate several unique functions for each of these caspases during cell death. Specific inhibition of caspase-9 allows for efficient release of cytochromec, but blocks changes in mitochondrial morphology and ROS production. We show that caspase-9 can cleave Bid into tBid at amino acid 59 and that this cleavage of Bid is required for ROS production following serum withdrawal. We also demonstrate that caspase-3-deficient MEFs are less sensitive to intrinsic cell death stimulation, yet have higher ROS production. In contrast, caspase-7-deficient MEFs are not resistance to intrinsic cell death, but remain attached to the ECM.

Taken together, these data suggest that caspase-9 is required for mitochondrial morphological changes and ROS production by cleaving and activating Bid into tBid. After activation by caspase-9, caspase-3 inhibits ROS production and is required for efficient execution of apoptosis, while effector caspase-7 is required for apoptotic cell detachment.

A hot new topic in medical treatment is the use of mesenchymal stem cells (MSC) in therapy. The low frequency of this subpopulation of stem cells in bone marrow (BM) necessitates their in vitro expansion prior to clinical use. We evaluated the effect of long term culture on the senescence of these cells.

The mean long term culture was 118 days and the mean passage number was 9. The average number of PD decreased from 7.7 to 1.2 in the 10th passage. The mean telomere length decreased from 9.19 Kbp to 8.7 kbp in the 9th passage. Differentiation potential dropped from the 6th passage on. The culture's morphological abnormalities were typical of the Hayflick model of cellular aging.

We believe that MSC enter senescence almost undetectably from the moment of in vitro culturing. Simultaneously these cells are losing their stem cell characteristics. Therefore, it is much better to consider them for cell and gene therapy early on.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P ₂] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)P ₂ also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)P ₂ levels in living cells is critical to understanding the biology of PtdIns(4,5)P ₂. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)P ₂ imaging tools.

All of the yeast PH domains that have been previously shown to bind PtdIns(4,5)P ₂ showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)P ₂. None of these domains showed any advantage over the PLCδ1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)P ₂ binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P₃. Detailed comparison of the Tubby and PLCδ1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)P ₂ and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.

These results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)P ₂ changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCδ1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P₃ binding may compromise its accuracy to measure PtdIns(4,5)P ₂changes. The Tubby domain is more accurate to report on PtdIns(4,5)P ₂ but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)P ₂ levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)P ₂.

Potential therapeutic use of mesenchymal stem cells (MSCs) is likely to require large-scale in vitro expansion of the cells before transplantation. MSCs from adipose tissue can be cultured extensively until senescence. However, little is known on the differentiation potential of adipose stem cells (ASCs) upon extended culture and on associated epigenetic alterations. We examined the adipogenic differentiation potential of clones of human ASCs in early passage culture and upon senescence, and determined whether senescence was associated with changes in adipogenic promoter DNA methylation.

ASC clones cultured to senescence display reduced adipogenic differentiation capacity in vitro, on the basis of limited lipogenesis and reduced transcriptional upregulation of FABP4 and LPL, two adipogenic genes, while LEP and PPARG2 transcription remains unaffected. In undifferentiated senescent cells, PPARG2 and LPL expression is unaltered, whereas LEP and FABP4 transcript levels are increased but not in all clones. Bisulfite sequencing analysis of DNA methylation reveals overall relative stability of LEP, PPARG2, FABP4 and LPL promoter CpG methylation during senescence and upon differentiation. Mosaicism in methylation profiles is maintained between and within ASC clones, and any CpG-specific methylation change detected does not necessarily relate to differentiation potential. One exception to this contention is CpG No. 21 in the LEP promoter, whose senescence-related methylation may impair upregulation of the gene upon adipogenic stimulation.

Senescent ASCs display reduced in vitro differentiation ability and transcriptional activation of adipogenic genes upon differentiation induction. These restrictions, however, cannot in general be attributed to specific changes in DNA methylation at adipogenic promoters. There also seems to be a correlation between CpGs that are hypomethylated and important transcription factor binding sites.

Epithelial to Mesenchymal Transition (EMT) induced by Transforming Growth Factor-β (TGF-β) is an important cellular event in organogenesis, cancer, and organ fibrosis. The process to reverse EMT is not well established. Our purpose is to define signaling pathways and transcription factors that maintain the TGF-β-induced mesenchymal state.

Inhibitors of five kinases implicated in EMT, TGF-β Type I receptor kinase (TβRI), p38 mitogen-activated protein kinase (p38 MAPK), MAP kinase kinase/extracellular signal-regulated kinase activator kinase (MEK1), c-Jun NH-terminal kinase (JNK), and Rho kinase (ROCK), were evaluated for reversal of the mesenchymal state induced in renal tubular epithelial cells. Single agents did not fully reverse EMT as determined by cellular morphology and gene expression. However, exposure to the TβRI inhibitor SB431542, combined with the ROCK inhibitor Y27632, eliminated detectable actin stress fibers and mesenchymal gene expression while restoring epithelial E-cadherin and Kidney-specific cadherin (Ksp-cadherin) expression. A second combination, the TβRI inhibitor SB431542 together with the p38 MAPK inhibitor SB203580, was partially effective in reversing EMT. Furthermore, JNK inhibitor SP600125 inhibits the effectiveness of the TβRI inhibitor SB431542 to reverse EMT. To explore the molecular basis underlying EMT reversal, we also targeted the transcriptional repressors ZEB1 and ZEB2/SIP1. Decreasing ZEB1 and ZEB2 expression in mouse mammary gland cells with shRNAs was sufficient to up-regulate expression of epithelial proteins such as E-cadherin and to re-establish epithelial features. However, complete restoration of cortical F-actin required incubation with the ROCK inhibitor Y27632 in combination with ZEB1/2 knockdown.

We demonstrate that reversal of EMT requires re-establishing both epithelial transcription and structural components by sustained and independent signaling through TβRI and ROCK. These findings indicate that combination small molecule therapy targeting multiple kinases may be necessary to reverse disease conditions.

Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions.

We have quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to mis-calculation of migration rates of up to 410%. In order to provide for objective measurements of cell migration rates, we have employed multi-target tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high throughput screening of video sequences of unstained living cells.

We demonstrate that our automatic multi target tracking system identifies cell objects, follows individual cells and computes migration rates with high precision, clearly outperforming manual procedures.

The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation.

Using transcriptional microarray analysis, we found that expression of 6 of the NETs significantly increases during myoblast differentiation. We confirmed these results using quantitative RT-PCR, and furthermore, found that all 6 NETs are expressed at high levels in adult mouse skeletal muscle relative to 9 other tissues examined. Using epitope-tagged cDNAs, we determined that the 5 NETs we could analyze (NETs 9, 25, 32, 37 and 39) all target to the nuclear envelope in C2C12 cells. Furthermore, the 3 NETs that we could analyze by immunoblotting were highly enriched in nuclear envelopes relative to microsomal membranes purified from mouse liver. Database searches showed that 4 of the 6 up-regulated NETs contain regions of homology to proteins previously linked to signaling.

This work identified 6 NETs that are predicted to have important functions in muscle development and/or maintenance from their expression patterns during myoblast differentiation and in mouse tissues. We confirmed that 5 of these NETs are authentic nuclear envelope proteins. Four members of this group have potential signaling functions at the NE, based on their sequence homologies.