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Biomedical subjects

Thomas M Roberts

Publications and source records attributed to Thomas M Roberts.

At least 19 recordsLinked to original sources
PubMed2007

Reconstitution in vitro of MSP-based filopodium extension in nematode sperm.

The major sperm protein (MSP) motility system in nematode sperm is best known for propelling the movement of mature sperm, where it has taken over the role usually played by actin in amoeboid cell motility. However, MSP filaments also drive the extension of filopodia, transient organelles composed of a core bundle of MSP filaments, that form in the late in sperm development but are not found on crawling cells. We have reconstituted filopodial extension in vitro whereby thin bundles of MSP filaments, each enveloped by a membrane sheath at their growing end, elongated at rates up to 17 microm/min. These bundles often exceeded 500 microm in length but were comprised of filaments only 1 microm long. The reconstituted filopodia assembled in the same cell-free sperm extracts that produced MSP fibers, robust meshworks of filaments that exhibit the same organization and dynamics as the lamellipodial filament system that propels sperm movement. The filopodia and fibers that assembled in vitro both had a membranous structure at their growing end, shared four MSP accessory proteins, and responded identically to agents that alter MSP-based motility by modulating protein phosphorylation. However, filopodia grew three- to four-fold faster than fibers. The reconstitution of filopodial extension shows that, like the actin cytoskeleton, MSP filaments can adopt two architectures, bundles and meshworks, each capable of pushing against membranes to generate protrusion. The reconstitution of both forms of motility in the same in vitro system provides a promising avenue for understanding how the forces for membrane protrusion are produced.

Actins↗
PubMed2006

PI3 kinases in cancer: from oncogene artifact to leading cancer target.

At the time of its discovery in the 1980s, the lipid kinase activity found associated with certain viral oncoproteins, which we now know to originate from the class IA phosphatidylinositide 3-kinases (PI3Ks), was thought by many to be a purification artifact. Subsequent work identified these enzymes as key regulators of cell signaling pathways that control various cellular processes including proliferation, motility, survival, and apoptosis. It is now clear that the PI3K pathway is activated by genetic or epigenetic alterations in a large proportion of human tumors, and a search for small molecule inhibitors of PI3K activity is currently a major effort of the pharmaceutical industry. However, because of the importance of PI3Ks in normal physiology, the road to therapeutics might not be smooth and could require further dissection of PI3K signaling pathways. In particular, it may be important to distinguish among the roles of the various isoforms of class 1A PI3K in both normal physiology and tumorigenesis.

Animals↗
PubMed2006

The p110alpha isoform of PI3K is essential for proper growth factor signaling and oncogenic transformation.

Growth factor signaling is mediated through Class IA phosphatidylinositol 3-kinases (PI3Ks). Among this class of enzymes, only p110alpha, encoded by the PIK3CA gene, has been found to be mutant in human cancers. To determine the specific functions of p110alpha, we generated mice carrying a conditionally targeted allele of the PIK3CA gene. Here, we report that PIK3CA-knockout mouse embryonic fibroblasts are deficient in cellular signaling in response to various growth factors, unable to differentiate into adipocytes, and resistant to oncogenic transformation induced by a variety of oncogenic receptor tyrosine kinases, indicating a fundamental role for p110alpha in these biological processes.

Adipocytes↗
PubMed2006

PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum.

Anti-apoptotic activity of BCL-2 is mediated by phosphorylation at the endoplasmic reticulum (ER), but how this phosphorylation is regulated and the mechanism(s) by which it regulates apoptosis are unknown. We purified macromolecular complexes containing BCL-2 from ER membranes and found that BCL-2 co-purified with the main two subunits of the serine/threonine phosphatase, PP2A. The association of endogenous PP2A and BCL-2 at the ER was verified by co-immunoprecipitation and microcystin affinity purification. Knock down or pharmacological inhibition of PP2A caused degradation of phosphorylated BCL-2 and led to an overall reduction in BCL-2 levels. We found that this degradation was due to the action of the proteasome acting selectively at the ER. Conversely, overexpression of PP2A caused elevation in endogenous BCL-2. Most importantly, we found that PP2A knock down sensitized cells to several classes of death stimuli (including ER stress), but this effect was abolished in a genetic background featuring knock in of a non-phosphorylatable BCL-2 allele. These studies support the hypothesis that PP2A-mediated dephosphorylation of BCL-2 is required to protect BCL-2 from proteasome-dependent degradation, affecting resistance to ER stress.

Amino Acid Sequence↗
PubMed2006

Human tumor mutants in the p110alpha subunit of PI3K.

The PI3K-Akt pathway is frequently upregulated in human tumors. Recently, somatic mutations of PIK3CA, encoding p110alpha catalytic subunit of Class IA PI3Ks, have been found in various cancers. The two most common types of p110alpha mutants, those in the helical and kinase domains, have been shown to be very potent in Akt activation and oncogenic transformation by several groups. Notably these common mutations may not enhance recruitment of p110alpha to the plasma membrane where its substrates are located. We have investigated the effect of membrane localization on common PIK3CA tumor mutants via myristoylation. In addition we have studied a third class of less frequent mutants in the p85-binding domain, in an attempt to gain insight into p85's inhibitory effect on p110alpha. This article briefly reviews and extends the literature on mutant forms of p110alpha.

Class I Phosphatidylinositol 3-Kinases↗
PubMed2006

Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.

We examined the role of angiogenesis and the need for receptor signaling using chemical inhibition of the vascular endothelial growth factor receptor in the adult zebrafish tail fin. Using a small-molecule inhibitor, we were able to exert precise control over blood vessel regeneration. An angiogenic limit to tissue regeneration was determined, as avascular tissue containing skin, pigment, neuronal axons and bone precursors could regenerate up to about 1 mm. This indicates that tissues can regenerate without direct interaction with endothelial cells and at a distance from blood supply. We also investigated whether the effects of chemical inhibition could be enhanced in zebrafish vascular mutants. We found that adult zebrafish, heterozygous for a mutation in the critical receptor effector phospholipase Cgamma1, show a greater sensitivity to chemical inhibition. This study illustrates the utility of the adult zebrafish as a new model system for receptor signaling and chemical biology.

Angiogenesis Inhibitors↗
PubMed2005

The oncogenic properties of mutant p110alpha and p110beta phosphatidylinositol 3-kinases in human mammary epithelial cells.

The PIK3CA gene encoding the p110alpha subunit of Class IA phosphatidylinositol 3-kinases (PI3Ks) is frequently mutated in human tumors. Mutations in the PIK3CB gene encoding p110beta, the only other widely expressed Class IA PI3K, have not been reported. We compared the biochemical activity and transforming potential of mutant forms of p110alpha and p110beta in a human mammary epithelial cell system. The two most common tumor-derived alleles of p110alpha, H1047R and E545K, potently activated PI3K signaling. Human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. We also examined a third class of mutations in p110alpha, those in the p85-binding domain. A representative tumor-derived p85-binding-domain mutant R38H showed modestly reduced p85 binding and weakly activated PI3K/Akt signaling. In contrast, a deletion mutant lacking the entire p85-binding domain efficiently activated PI3K signaling. When we constructed in p110beta a mutation homologous to the E545K allele of p110alpha, the resulting p110beta mutant was only weakly activated and allowed minimal soft-agar growth. However, a gene fusion of p110beta with the membrane anchor from c-Src was highly active and transforming in both soft-agar and orthotopic nude mouse assays. Thus, although introduction of activating mutations from p110alpha at the corresponding sites in p110beta failed to render the enzyme oncogenic in human cells, the possibility remains that other mutations might activate the beta isoform.

Alleles↗
PubMed2005

A genetic screen for candidate tumor suppressors identifies REST.

Tumorigenesis is a multistep process characterized by a myriad of genetic and epigenetic alterations. Identifying the causal perturbations that confer malignant transformation is a central goal in cancer biology. Here we report an RNAi-based genetic screen for genes that suppress transformation of human mammary epithelial cells. We identified genes previously implicated in proliferative control and epithelial cell function including two established tumor suppressors, TGFBR2 and PTEN. In addition, we uncovered a previously unrecognized tumor suppressor role for REST/NRSF, a transcriptional repressor of neuronal gene expression. Array-CGH analysis identified REST as a frequent target of deletion in colorectal cancer. Furthermore, we detect a frameshift mutation of the REST gene in colorectal cancer cells that encodes a dominantly acting truncation capable of transforming epithelial cells. Cells lacking REST exhibit increased PI(3)K signaling and are dependent upon this pathway for their transformed phenotype. These results implicate REST as a human tumor suppressor and provide a novel approach to identifying candidate genes that suppress the development of human cancer.

Cell Line, Tumor↗
PubMed2005

Structure of MFP2 and its function in enhancing MSP polymerization in Ascaris sperm amoeboid motility.

The simplicity and specialization of the cell motility machinery of Ascaris sperm provides a powerful system in which to probe the basic molecular mechanism of amoeboid cell motility. Although Ascaris sperm locomotion closely resembles that seen in many other types of crawling cell, movement is generated by modulation of a cytoskeleton based on the major sperm protein (MSP) rather than the actin present in other cell types. The Ascaris motility machinery can be studied conveniently in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibres constructed from bundles of MSP filaments. In addition to ATP, MSP and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins to orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. One of these proteins, MFP2, accelerates the rate of movement in this assay. Here, we describe crystal structures of two isoforms of MFP2 and show that both are constructed from two domains that have the same fold based on a novel, compact beta sheet arrangement. Patterns of conservation observed in a structure-based analysis of MFP2 sequences from different nematode species identified regions that may be putative functional interfaces involved both in interactions between MFP2 domains and also with other components of the sperm motility machinery. Analysis of the growth of fibres in vitro in the presence of added MFP2 indicated that MFP2 increases the rate of locomotion by enhancing the effective rate of MSP filament polymerization. This observation, together with the structural data, suggests that MFP2 may function in a manner analogous to formins in actin-based motility.

Amino Acid Sequence↗
PubMed2005

Preparing to move: assembly of the MSP amoeboid motility apparatus during spermiogenesis in Ascaris.

We exploited the rapid, inducible conversion of non-motile Ascaris spermatids into crawling spermatozoa to examine the pattern of assembly of the MSP motility apparatus that powers sperm locomotion. In live sperm, the first detectable motile activity is the extension of spikes and, later, blebs from the cell surface. However, examination of cells by EM revealed that the formation of surface protrusions is preceded by assembly of MSP filament tails on the membranous organelles in the peripheral cytoplasm. These organelle-associated filament meshworks assemble within 30 sec after induction of spermiogenesis and persist until the membranous organelles are sequestered into the cell body when the lamellipod extends. The filopodia-like spikes, which are packed with bundles of filaments, extend and retract rapidly but last only a few seconds before giving way to, or converting into, blebs. Coalescence of these blebs, each supported by a dense mesh of filaments, often initiates lamellipod extension, which culminates in the formation of the robust, dynamic MSP fiber complexes that generate sperm motility. The same membrane phosphoprotein that orchestrates assembly of the fiber complexes at the leading edge of the lamellipod of mature sperm is also found at all sites of filament assembly during spermiogenesis. The orderly progression of steps that leads to construction of a functional motility apparatus illustrates the precise spatio-temporal control of MSP filament assembly in the developing cell and highlights the remarkable similarity in organization and plasticity shared by the MSP cytoskeleton and the actin filament arrays in conventional crawling cells.

Animals↗
PubMed2005

Cytoskeleton dynamics powers nematode sperm motility.

Nematode sperm provide a simple and specialized system for studying the molecular mechanism of amoeboid cell motility. Locomotion is generated by the assembly dynamics of their cytoskeleton, which is based on the major sperm protein (MSP). Protrusive force is generated at the leading edge of the lamellipod by MSP filament formation and bundling, whereas the contractile force that drags the rearward cell body forward is generated by cytoskeleton disassembly. The dynamics of the system can be reconstituted in vitro using cell-free extracts of Ascaris sperm, in which vesicles derived from the leading edge of the cell can be either pushed or pulled. The addition of ATP to the cell-free extract initiates MSP filament polymerization and bundling immediately behind the vesicle, and the expansion of the resulting gel pushes the vesicle at rates comparable to those seen in living cells. In contrast, the addition of Yersinia tyrosine phosphatase generates depolymerization and gel contraction that pulls the vesicles. Overall, nematode sperm motility illustrates that cell locomotion can be generated by cytoskeletal dynamics alone without the use of myosin-like motor proteins.

Actins↗
PubMed2005

Membrane insertion by anthrax protective antigen in cultured cells.

The enzymatic moieties of anthrax toxin enter the cytosol of mammalian cells via a pore in the endosomal membrane formed by the protective antigen (PA) moiety. Pore formation involves an acidic pH-induced conformational rearrangement of a heptameric precursor (the prepore), in which the seven 2beta2-2beta3 loops interact to generate a 14-strand transmembrane beta-barrel. To investigate this model in vivo, we labeled PA with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) at cysteine residues introduced into the 2beta2-2beta3 loop. Each labeled PA was bound to CHO cells, and NBD fluorescence was monitored over time in stirred cell suspensions or by confocal microscopy. A strong increase was observed with NBD at positions 305, 307, 309, and 311, sites where side chains are predicted to face the bilayer, and little change was seen at residues 304, 306, 308, 310, and 312, sites where side chains are predicted to face the pore lumen. The increase at position 305 was inhibited by membrane-restricted quenchers, low temperature, or various reagents known to affect toxin action. Of the 24 NBD attachment sites examined, all but three gave results qualitatively consistent with the beta-barrel model. Besides supporting the beta-barrel model of membrane insertion, our results describe the time course of insertion and identify PA residues where NBD gives a strong signal upon membrane insertion in vivo.

4-Chloro-7-nitrobenzofurazan↗
PubMed2004

Androgen-induced differentiation and tumorigenicity of human prostate epithelial cells.

Androgen ablation is the primary treatment modality for patients with metastatic prostate cancer; however, the role of androgen receptor signaling in prostate cancer development remains enigmatic. Using a series of genetically defined immortalized and tumorigenic human prostate epithelial cells, we found that introduction of the androgen receptor induced differentiation of transformed prostate epithelial cells to a luminal phenotype reminiscent of organ-confined prostate cancer when placed in the prostate microenvironment. Moreover, androgen receptor expression converted previously androgen-independent, tumorigenic prostate epithelial cells into cells dependent on testosterone for tumor formation. These observations indicate that androgen receptor expression is oncogenic and addictive for the human prostate epithelium.

Androgens↗
PubMed2004

The serine/threonine kinase cyclin G-associated kinase regulates epidermal growth factor receptor signaling.

Cyclin G-associated kinase (GAK) is a serine/threonine kinase that features high homology outside its kinase domain with auxilin. Like auxilin, GAK has been shown to be a cofactor for uncoating clathrin vesicles in vitro. We investigated epidermal growth factor (EGF) receptor-mediated signaling in cells, in which GAK is down-regulated by small hairpin RNAs. Here, we report that down-regulation of GAK by small hairpin RNA has two pronounced effects on EGF receptor signaling: (i) the levels of receptor expression and tyrosine kinase activity go up by >50-fold; and (ii) the spectrum of downstream signaling is significantly changed. One very obvious result is a large increase in the levels of activated extracellular signal-regulated kinase 5 and Akt. These two effects of GAK down-regulation result from, at least in part, alterations in receptor trafficking, the most striking of which is the persistence of EGF receptor in altered cellular compartment along with activated extracellular signal-regulated kinase 5. The alterations resulting from GAK down-regulation can have distinctive biological consequences: In CV1P cells, down-regulation of GAK results in outgrowth of cells in soft agar, raising the possibility that loss of GAK function may promote tumorigenesis.

Cell Division↗
PubMed2004

Simian virus 40 large T antigen targets the spindle assembly checkpoint protein Bub1.

The mitotic spindle checkpoint protein Bub1 has been found to be mutated at low frequency in certain human cancers characterized by aneuploidy. Simian virus 40 large T antigen efficiently immortalizes rodent cells and occasionally transforms them to tumorigenicity. T antigen can also cause genomic instability, inducing chromosomal aberrations and aneuploidy. Here, we report an interaction between Bub1 and T antigen. T antigen coimmunoprecipitates with endogenous Bub1 and Bub3, another component of the spindle checkpoint complex. Genetic analysis demonstrates that the interaction of T antigen with Bub1 is not required for immortalization but is closely correlated with transformation. T antigen induces an override of the spindle checkpoint dependent on Bub1 binding. This interaction with proteins of the spindle checkpoint machinery suggests another role for T antigen and provides insight into its ability to cause chromosomal aberrations, aneuploidy, and transformation.

Amino Acid Sequence↗
PubMed2004

Functional genetics and experimental models of human cancer.

Abundant evidence supports the hypothesis that cancer arises from normal cells through the stepwise accumulation of genetic mutations. The study of cells obtained from patients with cancer has identified numerous molecules and pathways that fundamentally contribute to malignant transformation; however, cancer cell lines are often difficult to isolate or maintain, and the cell lines that are available for experimentation represent only a small subset of late-stage human cancers. Recent work has elucidated the role of telomerase in regulating human cell lifespan and has enabled the development of new experimental systems to study human cancer. This review highlights the recent progress in combining genetic methods and primary human cells to understand the role of specific genes and pathways in cancer pathogenesis.

Animals↗