Showing papers in "Journal of Cell Science in 2001"

Molecular complexity and dynamics of cell-matrix adhesions

Abstract: Currently >50 proteins have been reported to be associated with focal contacts and related ECM adhesions. Most of these contain multiple domains through which they can interact with different molecular partners, potentially forming a dense and heterogeneous protein network at the cytoplasmic faces of the adhesion site. The molecular and structural diversity of this 'submembrane plaque' is regulated by a wide variety of mechanisms, including competition between different partner proteins for the same binding sites, interactions triggered or suppressed by tyrosine phosphorylation, and conformational changes in component proteins, which can affect their reactivity. Indeed, integrin-mediated adhesions can undergo dynamic changes in structure and molecular properties from dot-like focal complexes to stress-fiber-associated focal contacts, which can further 'mature' to form fibronectin-bound fibrillar adhesions. These changes are driven by mechanical force generated by the actin- and myosin-containing contractile machinery of the cells, or by external forces applied to the cells, and regulated by matrix rigidity.

Rho GTPases and cell migration.

Abstract: Cell migration involves dynamic and spatially regulated changes to the cytoskeleton and cell adhesion. The Rho GTPases play key roles in coordinating the cellular responses required for cell migration. Recent research has revealed new molecular links between Rho family proteins and the actin cytoskeleton, showing that they act to regulate actin polymerization, depolymerization and the activity of actin-associated myosins. In addition, studies on integrin signalling suggest that the substratum continuously feeds signals to Rho proteins in migrating cells to influence migration rate. There is also increasing evidence that Rho proteins affect the organization of the microtubule and intermediate filament networks and that this is important for cell migration.

PKB/Akt: a key mediator of cell proliferation, survival and insulin responses?

Abstract: The serine/threonine protein kinase PKB (also known as Akt) is thought to be a key mediator of signal transduction processes. The identification of PKB substrates and the role PKB phosphorylation plays in regulating these molecules have been a major focus of research in recent years. A recently developed motif-profile scoring algorithm that can be used to scan the genome for potential PKB substrates is therefore a useful tool, although additional considerations, such as the evolutionary conservation of the phosphorylation site, must also be taken into account. Recent evidence indicates that PKB plays a key role in cancer progression by stimulating cell proliferation and inhibiting apoptosis and is also probably a key mediator of insulin signalling. These findings indicate that PKB is likely to be a hot drug target for the treatment of cancer, diabetes and stroke. There are, however, a number of pitfalls of methodologies currently employed to study PKB function, and therefore caution should be used in interpretation of such experiments.

p300/CBP proteins: HATs for transcriptional bridges and scaffolds

Abstract: p300/CBP transcriptional co-activator proteins play a central role in co-ordinating and integrating multiple signal-dependent events with the transcription apparatus, allowing the appropriate level of gene activity to occur in response to diverse physiological cues that influence, for example, proliferation, differentiation and apoptosis. p300/CBP activity can be under aberrant control in human disease, particularly in cancer, which may inactivate a p300/CBP tumour-suppressor-like activity. The transcription regulating-properties of p300 and CBP appear to be exerted through multiple mechanisms. They act as protein bridges, thereby connecting different sequence-specific transcription factors to the transcription apparatus. Providing a protein scaffold upon which to build a multicomponent transcriptional regulatory complex is likely to be an important feature of p300/CBP control. Another key property is the presence of histone acetyltransferase (HAT) activity, which endows p300/CBP with the capacity to influence chromatin activity by modulating nucleosomal histones. Other proteins, including the p53 tumour suppressor, are targets for acetylation by p300/CBP. With the current intense level of research activity, p300/CBP will continue to be in the limelight and, we can be confident, yield new and important information on fundamental processes involved in transcriptional control.

Identification of essential genes in cultured mammalian cells using small interfering RNAs.

Abstract: We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both beta- and gamma-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype. Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.

The splice variants of vascular endothelial growth factor (VEGF) and their receptors.

Abstract: Vascular endothelial growth factor (VEGF) is a secreted mitogen highly specific for cultured endothelial cells. In vivo VEGF induces microvascular permeability and plays a central role in both angiogenesis and vasculogenesis. VEGF is a promising target for therapeutic intervention in certain pathological conditions that are angiogenesis dependent, most notably the neovascularisation of growing tumours. Through alternative mRNA splicing, a single gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. Two VEGF receptor tyrosine kinases (VEGFRs) have been identified, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 seems to mediate almost all observed endothelial cell responses to VEGF, whereas roles for VEGFR-1 are more elusive. VEGFR-1 might act predominantly as a ligand-binding molecule, sequestering VEGF from VEGFR-2 signalling. Several isoform-specific VEGF receptors exist that modulate VEGF activity. Neuropilin-1 acts as a co-receptor for VEGF(165), enhancing its binding to VEGFR-2 and its bioactivity. Heparan sulphate proteoglycans (HSPGs), as well as binding certain VEGF isoforms, interact with both VEGFR-1 and VEGFR-2. HSPGs have a wide variety of functions, such as the ability to partially restore lost function to damaged VEGF(165) and thereby prolonging its biological activity.

Smad regulation in TGF-beta signal transduction.

Abstract: Smad proteins transduce signals from transforming growth factor-beta (TGF-beta) superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. Phosphorylation of receptor-activated Smads (R-Smads) leads to formation of complexes with the common mediator Smad (Co-Smad), which are imported to the nucleus. Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. Alternatively, nuclear R-Smads associate with ubiquitin ligases and promote degradation of transcriptional repressors, thus facilitating target gene regulation by TGF-beta. Smads themselves can also become ubiquitinated and are degraded by proteasomes. Finally, the inhibitory Smads (I-Smads) block phosphorylation of R-Smads by the receptors and promote ubiquitination and degradation of receptor complexes, thus inhibiting signalling.

Control of mitochondrial morphology by a human mitofusin

Abstract: Although changes in mitochondrial size and arrangement accompany both cellular differentiation and human disease, the mechanisms that mediate mitochondrial fusion, fission and morphogenesis in mammalian cells are not understood. We have identified two human genes encoding potential mediators of mitochondrial fusion. The mitofusins (Mfn1 and Mfn2) are homologs of the Drosophila protein fuzzy onion (Fzo) that associate with mitochondria and alter mitochondrial morphology when expressed by transient transfection in tissue culture cells. An internal region including a predicted bipartite transmembrane domain (TM) is sufficient to target Mfn2 to mitochondria and requires hydrophobic residues within the TM. Co-expression of Mfn2 with a dominant interfering mutant dynamin-related protein (Drp1(K38A)) proposed to block mitochondrial fission resulted in long mitochondrial filaments and networks. Formation of mitochondrial filaments and networks required a wild-type Mfn2 GTPase domain, suggesting that the Mfn2 GTPase regulates or mediates mitochondrial fusion and that mitofusins and dynamin related GTPases play opposing roles in mitochondrial fusion and fission in mammals, as in yeast.

Mechanism and role of PDZ domains in signaling complex assembly.

Abstract: PDZ domains are protein-protein recognition modules that play a central role in organizing diverse cell signaling assemblies. These domains specifically recognize short C-terminal peptide motifs, but can also recognize internal sequences that structurally mimic a terminus. PDZ domains can therefore be used in combination to bind an array of target proteins or to oligomerize into branched networks. Several PDZ-domain-containing proteins play an important role in the transport, localization and assembly of supramolecular signaling complexes. Examples of such PDZ-mediated assemblies exist in Drosophila photoreceptor cells and at mammalian synapses. The predominance of PDZ domains in metazoans indicates that this highly specialized scaffolding module probably evolved in response to the increased signaling needs of multicellular organisms.

SH3 domains: complexity in moderation

Abstract: The SH3 domain is perhaps the best-characterized member of the growing family of protein-interaction modules. By binding with moderate affinity and selectivity to proline-rich ligands, these domains play critical roles in a wide variety of biological processes ranging from regulation of enzymes by intramolecular interactions, increasing the local concentration or altering the subcellular localization of components of signaling pathways, and mediating the assembly of large multiprotein complexes. SH3 domains and their binding sites have cropped up in many hundreds of proteins in species from yeast to man, which suggests that they provide the cell with an especially handy and adaptable means of bringing proteins together. The wealth of genetic, biochemical and structural information available provides an intimate and detailed portrait of the domain, serving as a framework for understanding other modular protein-interaction domains. Processes regulated by SH3 domains also raise important questions about the nature of specificity and the overall logic governing networks of protein interactions.

Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1

Abstract: The function of many transmembrane molecules can be altered by cleavage and subsequent release of their ectodomains. We have investigated ectodomain cleavage of the cell-cell adhesion and signal-transducing molecule E-cadherin. The E-cadherin ectodomain is constitutively shed from the surface of MCF-7 and MDCKts.srcC12 cells in culture. Release of the 80 kDa soluble E-cadherin fragment is stimulated by phorbol-12-myristate-13-acetate and is inhibited by overexpression of the tissue inhibitor of metalloproteinases-2. The metalloproteinases matrilysin and stromelysin-1 both cleave E-cadherin at the cell surface and release sE-CAD into the medium. The soluble E-cadherin fragment thus released inhibits E-cadherin functions in a paracrine way, as indicated by induction of invasion into collagen type I and inhibition of E-cadherin-dependent cell aggregation. Our results, therefore, suggest a novel mechanism by which metalloproteinases can influence invasion.

Phagocytosis and the actin cytoskeleton

Abstract: The process of engulfing a foreign particle - phagocytosis - is of fundamental importance for a wide diversity of organisms. From simple unicellular organisms that use phagocytosis to obtain their next meal, to complex metazoans in which phagocytic cells represent an essential branch of the immune system, evolution has armed cells with a fantastic repertoire of molecules that serve to bring about this complex event. Regardless of the organism or specific molecules concerned, however, all phagocytic processes are driven by a finely controlled rearrangement of the actin cytoskeleton. A variety of signals can converge to locally reorganise the actin cytoskeleton at a phagosome, and there are significant similarities and differences between different organisms and between different engulfment processes within the same organism. Recent advances have demonstrated the complexity of phagocytic signalling, such as the involvement of phosphoinostide lipids and multicomponent signalling complexes in transducing signals from phagocytic receptors to the cytoskeleton. Similarly, a wide diversity of 'effector molecules' are now implicated in actin-remodelling downstream of these receptors.

WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement

Abstract: Reorganization of cortical actin filaments plays critical roles in cell movement and pattern formation. Recently, the WASP and WAVE family proteins WASP and N-WASP, and WAVE1, WAVE2 and WAVE3 have been shown to regulate cortical actin filament reorganization in response to extracellular stimuli. These proteins each have a verprolin-homology (V) domain, cofilin-homology (C) domain and an acidic (A) region at the C-terminus, through which they activate the Arp2/3 complex, leading to rapid actin polymerization. N-WASP is usually present as an inactive form in which the VCA region is masked. Cooperative binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) exposes the VCA region, activating N-WASP. In addition to this activation mechanism, WISH also activates N-WASP independently of Cdc42 and PtdIns(4,5)P(2), by binding to the proline-rich region of N-WASP. N-WASP activation induces formation of filopodia in vivo. In contrast, the ubiquitously expressed form of WAVE2 is activated downstream of Rac, leading to formation of lamellipodia. In this case, IRSp53 transmits a signal from Rac to WAVE2 through formation of a ternary Rac-IRSp53-WAVE2 complex. Thus, N-WASP, which is activated downstream of Cdc42 or independently by WISH, induces formation of filopodia and WAVE2, which is activated via IRSp53 downstream of Rac, induces formation of lamellipodia.

The cadherin superfamily: diversity in form and function.

Abstract: Over recent years cadherins have emerged as a growing superfamily of molecules, and a complex picture of their structure and their biological functions is becoming apparent. Variation in their extracellular region leads to the large potential for recognition properties of this superfamily. This is demonstrated strikingly by the recently discovered FYN-binding CNR-protocadherins; these exhibit alternative expression of the extracellular portion, which could lead to distinct cell recognition in different neuronal populations, whereas their cytoplasmic part, and therefore intracellular interactions, is constant. Diversity in the cytoplasmic moiety of the cadherins imparts specificity to their interactions with cytoplasmic components; for example, classical cadherins interact with catenins and the actin filament network, desmosomal cadherins interact with catenins and the intermediate filament system and CNR-cadherins interact with the SRC-family kinase FYN. Recent evidence suggests that CNR-cadherins, 7TM-cadherins and T-cadherin, which is tethered to the membrane by a GPI anchor, all localise to lipid rafts, specialised cell membrane domains rich in signalling molecules. Originally thought of as cell adhesion molecules, cadherin superfamily molecules are now known to be involved in many biological processes, such as cell recognition, cell signalling, cell communication, morphogenesis, angiogenesis and possibly even neurotransmission.

Complexes of tetraspanins with integrins: more than meets the eye

Abstract: The transmembrane proteins of the tetraspanin superfamily are implicated in a diverse range of biological phenomena, including cell motility, metastasis, cell proliferation and differentiation. The tetraspanins are associated with adhesion receptors of the integrin family and regulate integrin-dependent cell migration. In cells attached to the extracellular matrix, the integrin-tetraspanin adhesion complexes are clustered into a distinct type of adhesion structure at the cell periphery. Various tetraspanins are associated with phosphatidylinositol 4-kinase and protein kinase C isoforms, and they may facilitate assembly of signalling complexes by tethering these enzymes to integrin heterodimers. At the plasma membrane, integrin-tetraspanin signalling complexes are partitioned into specific microdomains proximal to cholesterol-rich lipid rafts. A substantial fraction of tetraspanins colocalise with integrins in various intracellular vesicular compartments. It is proposed that tetraspanins can influence cell migration by one of the following mechanisms: (1) modulation of integrin signalling; (2) compartmentalisation of integrins on the cell surface; or (3) direction of intracellular trafficking and recycling of integrins.

Mechanisms of capacitative calcium entry

Abstract: Capacitative Ca(2+) entry involves the regulation of plasma membrane Ca(2+) channels by the filling state of intracellular Ca(2+) stores in the endoplasmic reticulum (ER). Several theories have been advanced regarding the mechanism by which the stores communicate with the plasma membrane. One such mechanism, supported by recent findings, is conformational coupling: inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) receptors in the ER may sense the fall in Ca(2+) levels through Ca(2+)-binding sites on their lumenal domains, and convey this conformational information directly by physically interacting with Ca(2+) channels in the plasma membrane. In support of this idea, in some cell types, store-operated channels in excised membrane patches appear to depend on the presence of both Ins(1,4,5)P(3) and Ins(1,4,5)P(3) receptors for activity; in addition, inhibitors of Ins(1,4,5)P(3) production that either block phospholipase C or inhibit phosphatidylinositol 4-kinase can block capacitative Ca(2+) entry. However, the electrophysiological current underlying capacitative Ca(2+) entry is not blocked by an Ins(1,4,5)P(3) receptor antagonist, and the blocking effects of a phospholipase C inhibitor are not reversed by the intracellular application of Ins(1,4,5)P(3). Furthermore, cells whose Ins(1,4,5)P(3) receptor genes have been disrupted can nevertheless maintain their capability to activate capacitative Ca(2+) entry channels in response to store depletion. A tentative conclusion is that multiple mechanisms for signaling capacitative Ca(2+) entry may exist, and involve conformational coupling in some cell types and perhaps a diffusible signal in others.

Phosphoinositide 3-kinase signalling pathways.

Abstract: Phosphoinositide 3-kinases (PI3Ks) phosphorylate the 3′-OH position of the inositol ring of inositol phospholipids, producing three lipid products: PtdIns(3)P, PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). These lipids bind to the pleckstrin homology (PH) domains of proteins and control the activity and subcellular localisation of a diverse array of signal transduction molecules. Three major classes of signalling molecule are regulated by binding of D-3 phosphoinositides to PH domains: guanine-nucleotide-exchange proteins for Ρ family GTPases, the TEC family tyrosine kinases such as BTK and ITK in B and T lymphocytes, respectively, and the AGC superfamily of serine/threonine protein kinases. These molecules are activated by a variety of extracellular stimuli and have been implicated in a wide range of cellular processes, including cell cycle progression, cell growth, cell motility, cell adhesion and cell survival.

Integrins and cell proliferation: regulation of cyclin-dependent kinases via cytoplasmic signaling pathways.

Abstract: Cell cycle progression in mammalian cells is strictly regulated by both integrin-mediated adhesion to the extracellular matrix and by binding of growth factors to their receptors. This regulation is mediated by G1 phase cyclin-dependent kinases (CDKs), which are downstream of signaling pathways under the integrated control of both integrins and growth factor receptors. Recent advances demonstrate a surprisingly diverse array of integrin-dependent signals that are channeled into the regulation of the G1 phase CDKs. Regulation of cyclin D1 by the ERK pathway may provide a paradigm for understanding how cell adhesion can determine cell cycle progression.

A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation

Abstract: Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.

Post-transcriptional gene silencing in plants

Abstract: Post-transcriptional gene silencing (PTGS) in plants is an RNA-degradation mechanism that shows similarities to RNA interference (RNAi) in animals. Indeed, both involve double-stranded RNA (dsRNA), spread within the organism from a localised initiating area, correlate with the accumulation of small interfering RNA (siRNA) and require putative RNA-dependent RNA polymerases, RNA helicases and proteins of unknown functions containing PAZ and Piwi domains. However, some differences are evident. First, PTGS in plants requires at least two genes--SGS3 (which encodes a protein of unknown function containing a coil-coiled domain) and MET1 (which encodes a DNA-methyltransferase)--that are absent in C. elegans and thus are not required for RNAi. Second, all Arabidopsis mutants that exhibit impaired PTGS are hypersusceptible to infection by the cucumovirus CMV, indicating that PTGS participates in a mechanism for plant resistance to viruses. Interestingly, many viruses have developed strategies to counteract PTGS and successfully infect plants--for example, by potentiating endogenous suppressors of PTGS. Whether viruses can counteract RNAi in animals and whether endogenous suppressors of RNAi exist in animals is still unknown.

Inhibition of neuronal maturation in primary hippocampal neurons from tau deficient mice.

Abstract: Conflicting evidence supports a role for τ as an essential neuronal cytoskeletal protein or as a redundant protein whose function can be fulfilled by other microtubule-associated proteins. To investigate the function of τ in axonogenesis, we created τ deficient mice by disrupting the TAU gene. The engineered mice do not express the τ protein, appear physically normal and are able to reproduce. In contrast to a previously reported τ knockout mouse, embryonic hippocampal cultures from τ deficient mice show a significant delay in maturation as measured by axonal and neuritic extensions. The classic technique of selectively enhancing axonal growth by growth on laminin substrates failed to restore normal neuronal maturation of τ knockout neurons. By mating human TAU-gene transgenic and τ knockout mice, we reconstituted τ-deficient neurons with human τ proteins and restored a normal pattern of axonal growth and neuronal maturation. The ability of human τ proteins to rescue τ-deficient mouse neurons confirms that τ expression affects the rate of neurite extension.

9Rgr; and Rac but not Cdc42 regulate endothelial cell permeability

Abstract: Endothelial permeability induced by thrombin and histamine is accompanied by actin stress fibre assembly and intercellular gap formation. Here, we investigate the roles of the R family GTPases Rho1, Rac1 and Cdc42 in regulating endothelial barrier function, and correlate this with their effects on F-actin organization and intercellular junctions. RhoA, Rac1 and Cdc42 proteins were expressed efficiently in human umbilical vein endothelial cells by adenovirus-mediated gene transfer. We show that inhibition of R prevents both thrombin- and histamine-induced increases in endothelial permeability and decreases in transendothelial resistance. Dominant-negative RhoA and a R kinase inhibitor, Y-27632, not only inhibit stress fibre assembly and contractility but also prevent thrombin- and histamine-induced disassembly of adherens and tight junctions in endothelial cells, providing an explanation for their effects on permeability. In contrast, dominant-negative Rac1 induces permeability in unstimulated cells and enhances thrombin-induced permeability, yet inhibits stress fibre assembly, indicating that increased stress fibre formation is not essential for endothelial permeability. Dominant-negative Cdc42 reduces thrombin-induced stress fibre formation and contractility but does not affect endothelial cell permeability or responses to histamine. These results demonstrate that R and Rac act in different ways to alter endothelial barrier function, whereas Cdc42 does not affect barrier function.

Tumor suppressor PTEN: modulator of cell signaling, growth, migration and apoptosis.

Abstract: PTEN (also known as MMAC-1 or TEP-1) is one of the most frequently mutated tumor suppressors in human cancer. It is also essential for embryonic development. PTEN functions primarily as a lipid phosphatase to regulate crucial signal transduction pathways; a key target is phosphatidylinositol 3,4,5-trisphosphate. In addition, it displays weak tyrosine phosphatase activity, which may downmodulate signaling pathways that involve focal adhesion kinase (FAK) or Shc. Levels of PTEN are regulated in embryos and adult organisms, and gene-targeting studies demonstrate that it has a crucial role in normal development. Functions for PTEN have been identified in the regulation of many normal cell processes, including growth, adhesion, migration, invasion and apoptosis. PTEN appears to play particularly important roles in regulating anoikis (apoptosis of cells after loss of contact with extracellular matrix) and cell migration. Gene targeting and transient expression studies have provided insight into the specific signaling pathways that regulate these processes. Characterization of the diverse signaling networks modulated by PTEN, as well as the regulation of PTEN concentration, enzymatic activity, and coordination with other phosphatases, should provide intriguing new insight into the biology of normal and malignant cells.

Effects of cytochalasin D and latrunculin B on mechanical properties of cells

Abstract: Actin microfilaments transmit traction and contraction forces generated within a cell to the extracellular matrix during embryonic development, wound healing and cell motility, and to maintain tissue structure and tone. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and tissues. Cytochalasin D and Latrunculin are commonly used reagents that, by different mechanisms, alter the state of actin polymerization or the organization of actin filaments. We have investigated the effect of a wide range of Cytochalasin D and Latrunculin B concentrations (from 40 pM to 10 microM) on the mechanical properties of the cells within fibroblast populated collagen matrices. Contractile force and dynamic stiffness were measured by uniaxial stress-strain testing. The range of effective concentrations of Cytochalasin D (200 pM-2 microM) was broader than that of Latrunculin B (20 nM-200 nM). Activating the cells by serum did not change the effective range of Cytochalasin D concentrations but shifted that of Latrunculin B upward by tenfold. Simple mathematical binding models based on the presumed mechanisms of action of Cytochalasin D and Latrunculin B simulated the concentration-dependent mechanical changes reasonably well. This study shows a strong dependence of the mechanical properties of cells and tissues on the organization and degree of polymerization of actin filaments.

Cell motility: can Rho GTPases and microtubules point the way?

Abstract: Migrating cells display a characteristic polarization of the actin cytoskeleton. Actin filaments polymerise in the protruding front of the cell whereas actin filament bundles contract in the cell body, which results in retraction of the cell's rear. The dynamic organization of the actin cytoskeleton provides the force for cell motility and is regulated by small GTPases of the Rho family, in particular Rac1, RhoA and Cdc42. Although the microtubule cytoskeleton is also polarized in a migrating cell, and microtubules are essential for the directed migration of many cell types, their role in cell motility is not well understood at a molecular level. Here, we discuss the potential molecular mechanisms for interplay of microtubules, actin and Rho GTPase signalling in cell polarization and motility. Recent evidence suggests that microtubules locally modulate the activity of Rho GTPases and, conversely, Rho GTPases might be responsible for the initial polarization of the microtubule cytoskeleton. Thus, microtubules might be part of a positive feedback mechanism that maintains the stable polarization of a directionally migrating cell.

The organisation and functions of local Ca(2+) signals.

Abstract: Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca(2+) to play a pivotal role in cell biology results from the facility that cells have to shape Ca(2+) signals in space, time and amplitude. To generate and interpret the variety of observed Ca(2+) signals, different cell types employ components selected from a Ca(2+) signalling 'toolkit', which comprises an array of homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca(2+) signals that suit their physiology. Recent studies have demonstrated the importance of local Ca(2+) signals in defining the specificity of the interaction of Ca(2+) with its targets. Furthermore, local Ca(2+) signals are the triggers and building blocks for larger global signals that propagate throughout cells.

TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-(kappa)B mediated induction of MT1-MMP.

Abstract: Tumor necrosis factor-alpha (TNF-(alpha)) is an important mediator during the inflammatory phase of wound healing. Excessive amounts of pro-inflammatory cytokines such as TNF-(alpha) are associated with inflammatory diseases including chronic wounds. Matrix metalloproteinases (MMPs) are involved in matrix re-modeling during wound healing, angiogenesis and tumor metastasis. As with pro-inflammatory cytokines, high levels of MMPs have been found in inflammatory states such as chronic wounds. In this report we relate these two phenomena. TNF-(alpha) stimulates secretion of active MMP-2, a type IV collagenase, in organ-cultured full-thickness human skin. This suggests a mechanism whereby excess inflammation affects normal wound healing. To investigate this observation at the cellular and molecular levels, we examined TNF-(alpha) mediated activation of pro-MMP-2, induction of MT1-MMP, and the intracellular signaling pathways that regulate the proteinase in isolated human dermal fibroblasts. We found that TNF-(alpha) substantially promoted activation of pro-MMP-2 in dermal fibroblasts embedded in type-I collagen. In marked contrast, collagen or TNF-(alpha) individually had little influence on the fibroblast-mediated pro-MMP-2 activation. One well-characterized mechanism for pro-MMP-2 activation is through a membrane type matrix metalloproteinase, such as MT1-MMP. We report that TNF-(alpha) significantly induced MT1-MMP at the mRNA and protein levels when the dermal fibroblasts were grown in collagen. Although the intracellular signaling pathway regulating mt1-mmp gene expression is still obscure, both TNF-(alpha) and collagen activate the NF-(kappa)B pathway. In this report we provide three sets of evidence to support a hypothesis that activation of NF-(kappa)B is essential to induce MT1-MMP expression in fibroblasts after TNF-(alpha) exposure. First, SN50, a peptide inhibitor for NF-(kappa)B nuclear translocation, simultaneously blocked the TNF-(alpha) and collagen mediated MT1-MMP induction and pro-MMP-2 activation. Secondly, TNF-(alpha) induced I(kappa)B to breakdown in fibroblasts within the collagen lattice, a critical step leading to NF-(kappa)B activation. Lastly, a consensus binding site for p65 NF-(kappa)B (TGGAGCTTCC) was found in the 5'-flanking region of human mt1-mmp gene. Based on these results and previous reports, we propose a model to explain TNF-(alpha) activation of MMP-2 in human skin. Activation of NF(kappa)B signaling in fibroblasts embedded in collagen induces mt1-mmp gene expression, which subsequently activates the pro-MMP-2. The findings provide a specific mechanism whereby TNF-(alpha) may affect matrix remodeling during wound healing and other physiological and pathological processes.

Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues.

Abstract: In search of vascular smooth muscle cell differentiation markers, we identified two genes encoding members of a new family of type II integral membrane proteins. Both are ubiquitously expressed, and tissue-specific alternative mRNA initiation and splicing generate at least two major isoforms of each protein, with the smaller isoforms being truncated at the N-terminus. We have named these proteins nesprin-1 and -2 for nuclear envelope spectrin repeat, as they are characterized by the presence of multiple, clustered spectrin repeats, bipartite nuclear localization sequences and a conserved C-terminal, single transmembrane domain. Transient transfection of EGFP-fusion expression constructs demonstrated their localization to the nuclear membrane with a novel C-terminal, TM-domain-containing sequence essential for perinuclear localization. Using antibodies to nesprin-1, we documented its colocalization with LAP1, emerin and lamins at the nuclear envelope, and immunogold labeling confirmed its presence at the nuclear envelope and in the nucleus where it colocalized with heterochromatin. Nesprin-1 is developmentally regulated in both smooth and skeletal muscle and is re-localized from the nuclear envelope to the nucleus and cytoplasm during C2C12 myoblast differentiation. These data and structural analogies with other proteins suggest that nesprins may function as ‘dystrophins of the nucleus’ to maintain nuclear organization and structural integrity.

Differential gene expression during capillary morphogenesis in 3D collagen matrices: regulated expression of genes involved in basement membrane matrix assembly, cell cycle progression, cellular differentiation and G-protein signaling.

Abstract: We have performed a screening analysis of differential gene expression using a defined in vitro model of human capillary tube formation. Gene array, differential display and cDNA library screening were used to identify both known and novel differentially expressed genes. Major findings include: the upregulation and functional importance of genes associated with basement membrane matrix assembly; the upregulation of growth factors, transcription factors, anti-apoptotic factors, markers of endothelial cell differentiation, JAK-STAT signalling molecules, adhesion receptors, proteinase inhibitors and actin regulatory proteins; and expression changes consistent with inhibition of cell cycle progression, increased cholesterol biosynthesis, decreased ubiquitin-proteasome mediated degradation, and activation of G-protein signaling pathways. Using DNA microarray analysis, the most induced genes at 8, 24 and 48 hours compared with those at 0 hours were jagged-1, stanniocalcin and angiopoietin-2, whereas the most repressed genes were connective tissue growth factor, fibulin-3 and RGS-5. In addition, the full length coding sequence of two novel regulated capillary morphogenesis genes (CMGs) are presented. CMG-1 encodes a predicted intracellular 65 kDa protein with coiled-coil domains. A CMG-1-green fluorescent protein (GFP) chimera was observed to target to an intracellular vesicular compartment. A second novel gene, CMG-2, was found to encode a predicted intracellular protein of 45 kDa containing a transmembrane segment and a CMG-2-GFP chimera was observed to target to the endoplasmic reticulum. A recombinant portion of CMG-2 was found to bind collagen type IV and laminin, suggesting a potential role in basement membrane matrix synthesis and assembly. These data further elucidate the genetic events regulating capillary tube formation in a 3D matrix environment.

Internalization of cholera toxin by different endocytic mechanisms

Abstract: The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-beta-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.