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The ability to develop animals that have human organs could save the lives of people waiting for transplants, but ethical issues still need to be faced. The ability to develop animals that have human organs could save the lives of people waiting for transplants, but ethical issues still need to be faced., Author(s): Liam Drew Author Affiliations: Credit: Markos Kay Artisitic representation of chimaeric cells The chimaera challenge It's 2036, and you have kidney failure. Until recently, this condition meant months or [...]

A rapidly growing collection of software tools is helping researchers to analyse multiple huge '-omics' data sets. A rapidly growing collection of software tools is helping researchers to analyse multiple huge '-omics' data sets., Author(s): Jeffrey M. Perkel Author Affiliations: Single-cell analysis enters the multiomics age Single-cell data (top) increasingly are being combined with spatial information (bottom). Credit: 10x Genomics Two sheets of visual [...]

The characterization of physical properties of cells such as their mass and stiffness has been of great interest and can have profound implications in cell biology, tissue engineering, cancer, and disease research. For example, the direct dependence of cell growth rate on cell mass for individual adherent human cells can elucidate the mechanisms underlying cell cycle progression. Here we develop an array of micro-electro-mechanical systems (MEMS) resonant mass sensors that can be used to directly measure the biophysical properties, mass, and growth rate of single adherent cells. Unlike conventional cantilever mass sensors, our sensors retain a uniform mass sensitivity over the cell attachment surface. By measuring the frequency shift of the mass sensors with growing (soft) cells and fixed (stiff) cells, and through analytical modeling, we derive the Young's modulus of the unfixed cell and unravel the dependence of the cell mass measurement on cell stiffness. Finally, we grew individual cells on the mass sensors and measured their mass for 50+ hours. Our results demonstrate that adherent human colon epithelial cells have increased growth rates with a larger cell mass, and the average growth rate increases linearly with the cell mass, at 3.25%/hr. Our sensitive mass sensors with a position-independent mass sensitivity can be coupled with microscopy for simultaneous monitoring of cell growth and status, and provide an ideal method to study cell growth, cell cycle progression, differentiation, and apoptosis. cell mechanics | cell division | bio-sensor doi/ 10.1073/pnas.1011365107

Murine hematopoietic blast colony-forming cells (BL-CFCs) are able to generate up to 30,000 progeny blast cells within 10 d in agar cultures. Contained in these populations are large numbers of lineage-committed progenitor cells in the granulocytic and macrophage lineages. Sequential analyses of blast colonies revealed that self-generation of BL-CFCs occurs but is surprisingly late in clonal expansion, as is the emergence of progenitor cells committed to megakaryocytic and eosinophil lineages. Self-generating BL-CFCs were highly enriched in [lineage.sup.-] [Kit.sup.+] [Sca1.sup.+] [CD34.sup.-] [Flt3R.sup.-] populations, and colonies generated by such cells contained colony-forming units--spleen and formed erythroid and lymphoid progeny in vivo. The data suggest the existence of a hierarchical structure in BL-CFC populations with at least a subset being cells assayable as colony-forming units-spleen. Because BL-CFCs can self-generate and are able to generate lymphoid and myeloid populations, BL-CFCs appear to be ideal cells in which to analyze the processes of self-generation and lineage commitment in clonal in vitro cultures. colony-forming units-spleen | megakaryocytes | eosinophils www.pnas.org/cgi/doi/ 10.1073/pnas.1011881107

Biological processes such as circadian rhythms, cell division, metabolism, and development occur as ordered sequences of events. The synchronization of these coordinated events is essential for proper cell function, and hence the determination of critical time points in biological processes is an important component of all biological investigations. In particular, such critical time points establish logical ordering constraints on subprocesses, impose prerequisites on temporal regulation and spatial compartmentalization, and situate dynamic reorganization of functional elements in preparation for subsequent stages. Thus, building temporal phenomenological representations of biological processes from genome-wide datasets is relevant in formulating biological hypotheses on: how processes are mechanistically regulated; how the regulations vary on an evolutionary scale, and how their inadvertent disregulation leads to a diseased state or fatality. This paper presents a general framework (GOALIE) to reconstruct temporal models of cellular processes from time-course gene expression data. We mathematically formulate the problem as one of optimally segmenting datasets into a succession of 'informative' windows such that time points within a window expose concerted clusters of gene action whereas time points straddling window boundaries constitute points of significant restructuring. We illustrate here how GOALIE successfully brings out the interplay between multiple yeast processes, inferred from combined experimental datasets for the cell cycle and the metabolic cycle. model building and model-checking | temporal data analysis | yeast cell cycle | yeast metabolic cycle | Kripke structures doi/10.1073/pnas.1006283107

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)- containing proteins involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial pathways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreich's ataxia) has established that communication exists between iron metabolism in the mit0chondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism. iron sulfur cluster | heine | Friedreich's ataxia | frataxin | sideroblastic anemia doi/ 10.1073/pnas.0912925107

Terminally differentiated cells in Drosophila melanogaster wings and eyes are largely resistant to proliferation upon deregulation of either E2F or cyclin E (CycE), but exogenous expression of both factors together can bypass cell cycle exit. In this study, we show this is the result of cooperation of cell cycle control mechanisms that limit E2F-CycE positive feedback and prevent cycling after terminal differentiation. Aberrant CycE activity after differentiation leads to the degradation of E2F activator complexes, which increases the proportion of CycE-resistant E2F repressor complexes, resulting in stable E2F target gene repression. If E2F-dependent repression is lost after differentiation, high anaphase-promoting complex/cyclosome (APC/C) activity degrades key E2F targets to limit cell cycle reentry. Providing both CycE and E2F activities bypasses exit by simultaneously inhibiting the APC/C and inducing a group of E2F target genes essential for cell cycle reentry after differentiation. These mechanisms are essential for proper development, as evading them leads to tissue outgrowths composed of dividing but terminally differentiated cells. doi/10.1083/jcb.200910006

Telomeres, the protein--DNA complexes at the ends of linear chromosomes, are protected and regulated by the shelterin molecules, the telomerase complex, and other accessory factors, among which is Apollo, a DNA repair factor of the [beta]-lactamase/[beta]-CASP family. Impaired telomere protection in humans causes dyskeratosis congenita and Hoyeraal--Hreidarsson (HH) syndrome, characterized by premature aging, bone marrow failure, and immunodeficiency. We identified a unique Apollo splice variant (designated Apollo-[DELTA]) in fibroblasts from a patient with HH syndrome. Apollo-[DELTA] generates a dominant negative form of Apollo lacking the telomeric repeat-binding factor homology (TRFH)-binding motif (TBM) required for interaction with the shelterin TRF2 at telomeres. Apollo-[DELTA] hampers the proper replication of telomeres, leading to major telomeric dysfunction and cellular senescence, but maintains its DNA interstrand cross-link repair function in the whole genome. These results identify Apollo as a crucial actor in telomere maintenance in vivo, independent of its function as a general DNA repair factor. telomeric repeat-binding factor homology-binding motif | TRF2 doi/ 10.1073/pnas.0914918107

Type V myosin (MyoV)-dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2-tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail-lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain. doi/ 10.1083/jcb.201002076

Integrins are fundamental to the control of protrusion and motility in adherent cells. However, the mechanisms by which specific members of this receptor family cooperate in signaling to cytoskeletal and adhesion dynamics are poorly understood. Here, we show that the loss of [beta]3 integrin in fibroblasts results in enhanced focal adhesion turnover and migration speed but impaired directional motility on both 2D and 3D matrices. These motility defects are coupled with an increased rate of actin-based protrusion. Analysis of downstream signaling events reveals that loss of [beta]3 integrin results in a loss of protein kinase A--dependent phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP). Dephosphorylated VASP in [beta]3-null cells is preferentially associated with Rap1-GTP--interacting adaptor molecule (RIAM) both in vitro and in vivo, which leads to enhanced formation of a VASP--RIAM complex at focal adhesions and subsequent increased binding of talin to [beta]1 integrin. These data demonstrate a novel mechanism by which [alpha]v[beta]3 integrin acts to locally suppress [beta]1 integrin activation and regulate protrusion, adhesion dynamics, and persistent migration. doi/10.1083/jcb.200912014

Although directed cellular migration facilitates the coordinated movement of cells during development and repair, the mechanisms regulating such migration remain poorly understood. Missing-in-metastasis (MIM) is a defining member of the inverse Bin/Amphiphysin/Rvs domain (I-BAR) subfamily of lipid binding, cytoskeletal regulators whose levels are altered in a number of cancers. Here, we provide the first genetic evidence that an I-BAR protein regulates directed cell migration in vivo. Drosophila MIM (dmim) is involved in Drosophila border cell migration, with loss of dmim function resulting in a lack of directional movement by the border cell cluster. In vivo endocytosis assays combined with genetic analyses demonstrate that the dmim product regulates directed cell movement by inhibiting endocytosis and antagonizing the activities of the CD2-associated protein/cortactin complex in these cells. These studies demonstrate that DMIM antagonizes pro-endocytic components to facilitate polarity and localized guidance cue sensing during directional cell migration. doi/10.1083/jcb.200910136

Precisely controlled growth and morphogenesis of developing epithelial tissues require coordination of multiple factors, including proliferation, adhesion, cell shape, and apoptosis. RhoA, a small GTPase, is known to control epithelial morphogenesis and integrity through its ability to regulate the cytoskeleton. In this study, we examine a less well-characterized RhoA function in cell survival. We demonstrate that the Drosophila melanogaster RhoA, Rho1, promotes apoptosis independently of Rho kinase through its effects on c-Jun N[H.sub.2]-terminal kinase (JNK) signaling. In addition, Rho1 forms a complex with Slipper (Slpr), an upstream activator of the JNK pathway. Loss of Moesin (Moe), an upstream regulator of Rho1 activity, results in increased levels of Rho1 at the plasma membrane and cortical accumulation of Slpr. Together, these results suggest that Rho1 functions at the cell cortex to regulate JNK activity and implicate Rho1 and Moe in epithelial cell survival. doi/10.1083/jcb.200912010

The formation of functional kinetochores requires the accurate assembly of a large number of protein complexes. The Hsp90--Sgt1 chaperone complex is important for this process; however, its targets are not conserved and its exact contribution to kinetochore assembly is unclear. Here, we show that human Hsp90--Sgt1 interacts with the Mis12 complex, a so-called keystone complex required to assemble a large fraction of the kinetochore. Inhibition of Hsp90 or Sgt1 destabilizes the Mis12 complex and delays proper chromosome alignment due to inefficient formation of microtubule-binding sites. Interestingly, coinhibition of Sgt1 and the SCF subunit, Skp1, increases Mis12 complexes at kinetochores and restores timely chromosome alignment but forms less-robust microtubule-binding sites. We propose that a balance of Mis12 complex assembly and turnover is required for the efficient and accurate assembly of kinetochore--microtubule binding sites. These findings support a novel role for Hsp90--Sgt1 chaperones in ensuring the fidelity of multiprotein complex assembly. doi/10.1083/jcb.200910036

The yeast Hsp70/40 system SSB--RAC (stress 70 B-ribosome-associated complex) binds to ribosomes and contacts nascent polypeptides to assist co-translational folding. In this study, we demonstrate that nascent polypeptide-associated complex (NAC), another ribosome-tethered system, is functionally connected to SSB-RAC and the cytosolic Hsp70 network. Simultaneous deletions of genes encoding NAC and SSB caused conditional loss of cell viability under protein-folding stress conditions. Furthermore, NAC mutations revealed genetic interaction with a deletion of Sse1, a nucleotide exchange factor regulating the cytosolic Hsp70 network. Cells lacking SSB or Sse1 showed protein aggregation, which is enhanced by additional loss of NAC; however, these mutants differ in their potential client repertoire. Aggregation of ribosomal proteins and biogenesis factors accompanied by a pronounced deficiency in ribosomal particles and translating ribosomes only occurs in ssb[DELTA] and nac[DELTA]ssb[DELTA] cells, suggesting that SSB and NAC control ribosome biogenesis. Thus, SSB--RAC and NAC assist protein folding and likewise have important functions for regulation of ribosome levels. These findings emphasize the concept that ribosome production is coordinated with the protein-folding capacity of ribosome-associated chaperones. doi/ 10.1083/jcb.200910074

Epithelial paracellular barrier function, determined primarily by tight junction permeability, is frequently disrupted in disease. In the intestine, barrier loss can be mediated by tumor necrosis factor ([alpha]) (TNF) signaling and epithelial myosin light chain kinase (MLCK) activation. However, TNF induces only limited alteration of tight junction morphology, and the events that couple structural reorganization to barrier regulation have not been defined. We have used in vivo imaging and transgenic mice expressing fluorescent-tagged occludin and ZO-1 fusion proteins to link occludin endocytosis to TNF-induced tight junction regulation. This endocytosis requires caveolin-1 and is essential for structural and functional tight junction regulation. These data demonstrate that MLCK activation triggers caveolin-1--dependent endocytosis of occludin to effect structural and functional tight junction regulation. doi/ 10.1083/jcb.200902153

The centrosomal protein, CDK5RAP2, is mutated in primary microcephaly, a neurodevelopmental disorder characterized by reduced brain size. The Drosophila melanogaster homologue of CDK5RAP2, centrosomin (Cnn), maintains the pericentriolar matrix (PCM) around centrioles during mitosis. In this study, we demonstrate a similar role for CDK5RAP2 in vertebrate cells. By disrupting two evolutionarily conserved domains of CDK5RAP2, CNN1 and CNN2, in the avian B cell line DT40, we find that both domains are essential for linking centrosomes to mitotic spindle poles. Although structurally intact, centrosomes lacking the CNN1 domain fail to recruit specific PCM components that mediate attachment to spindle poles. Furthermore, we show that the CNN1 domain enforces cohesion between parental centrioles during interphase and promotes efficient DNA damage--induced G2 cell cycle arrest. Because mitotic spindle positioning, asymmetric centrosome inheritance, and DNA damage signaling have all been implicated in cell fate determination during neurogenesis, our findings provide novel insight into how impaired CDK5RAP2 function could cause premature depletion of neural stem cells and thereby microcephaly. doi/10.1083/jcb.200912163

Patients with primary (AL) cardiac amyloidosis suffer from progressive cardiomyopathy with a median survival of less than 8 months and a 5-year survival of amyloid | cell death | heart failure | TAB1 | reactive oxygen species doi/10.1073/pnas.0912263107

The immune receptor signaling pathway is used by nonimmune cells, but the molecular adaptations that underlie its functional diversification are not known. Circulating platelets use the immune receptor homologue glycoprotein VI (GPVI) to respond to collagen exposed at sites of vessel injury. In contrast to immune cell responses, platelet activation must take place within seconds to successfully form thrombi in flowing blood. Here, we show that the GPVI receptor utilizes a unique intracellular proline-rich domain (PRD) to accelerate platelet activation, a requirement for efficient platelet adhesion to collagen under flow. The GPVI PRD specifically binds the Src-family kinase Lyn and directly activates it, presumably through SH3 displacement. In resting platelets, Lyn is constitutively bound to GPVI in an activated state and platelets lacking Lyn exhibit defective collagen adhesion like that of platelets with GPVI receptors lacking the PRD. These findings define a molecular priming mechanism that enables an immune-type receptor to adopt a hemostatic function. These studies also demonstrate that active kinases can constitutively associate with immune-type receptors without initiating signal transduction before receptor ligation, consistent with a recent molecular model of immune receptor signaling in which receptor ligation is required to bring active kinases to their receptor substrates. doi/10.1073/pnas.0906436106

High-energy inositol pyrophosphates, such as [IP.sup.7] (diphosphoinositol pentakisphosphate), can directly donate a [beta]-phosphate to a prephosphorylated serine residue generating pyrophosphorylated proteins. Here, we show that the [beta] subunit of AP-3, a clathrin-associated protein complex required for HIV-1 release, is a target of [IP.sub.7]-mediated pyrophosphoryiation. We have identified Kif3A, a motor protein of the kinesin superfamily, as an AP3B1-binding partner and demonstrate that Kif3A, like the AP-3 complex, is involved in an intracellular process required for HIV-1 Gag release. Importantly, [IP.sub.7]-mediated pyrophosphorylation of AP3B1 modulates the interaction with Kif3A and, as a consequence, affects the release of HIV-1 virus-like particles. This study identifies a cellular process that is regulated by [IP.sub.7]mediated pyrophosphorylation. phosphorylation | trafficking | kinesin doi/10.1073/pnas.0909176106

Dendritic cells (DC) are the professional antigen presenting cells (APC) that bridge the innate and adaptive immune system. Previously, in a CD11c/EYFP transgenic mouse developed to study DC functions, we anatomically mapped and phenotypically characterized a discrete population of [EYFP.sup.+] cells within the microglia that we termed brain dendritic cells (bDC). In this study, we advanced our knowledge of the function of these cells in the CD11c/EYFP transgenic mouse and its chimeras, using acute stimuli of stereotaxically inoculated IFN[gamma] or IL-4 into the CNS. The administration of IFN[gamma], increased the number of [EYFP.sup.+]bDC but did not recruit peripheral DC into the CNS. IFN[gamma], but not IL-4, upregulated the expression levels of major histocompatibility class II (MHC-II). In addition, IFN[gamma]-activated [EYFP.sup.+]bDC induced antigen-specific naive CD4 T cells to proliferate and secrete Th1/Th17 cytokines. Activated bDC were also able to stimulate naive CD8 T cells. Collectively, these data reveal the Th1 cytokine IFN[gamma], but not the Th2 cytokine IL4, induces bDC to up-regulate MHC-II and become competent APC. doi/10.1073/pnas.0911509106

All organisms need to ensure that no DNA segments are re-replicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a 'permissive' heterotrimer and an 'inhibitory' heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states. solution structure | X-ray structure | pre-RC | cell cycle doi/10.1073/pnas.0905281106

Defects in nuclear morphology often correlate with the onset of disease, including cancer, progeria, cardiomyopathy, and muscular dystrophy. However, the mechanism by which a cell controls its nuclear shape is unknown. Here, we use adhesive micropatterned surfaces to control the overall shape of fibroblasts and find that the shape of the nucleus is tightly regulated by the underlying cell adhesion geometry. We found that this regulation occurs through a dome-like actin cap that covers the top of the nucleus. This cap is composed of contractile actin filament bundles containing phosphorylated myosin, which form a highly organized, dynamic, and oriented structure in a wide variety of cells. The perinuclear actin cap is specifically disorganized or eliminated by inhibition of actomyosin contractility and rupture of the LINC complexes, which connect the nucleus to the actin cap. The organization of this actin cap and its nuclear shape-determining function are disrupted in cells from mouse models of accelerated aging (progeria) and muscular dystrophy with distorted nuclei caused by alterations of A-type lamins. These results highlight the interplay between cell shape, nuclear shape, and cell adhesion mediated by the perinuclear actin cap. LINC complexes I nucleus www.pnas.org/cgi/doi/10.1073/pnas.0908686106

Great arteries, as well as lungs and skin, contain elastic fibers as important components to maintain their physiological functions. Although recent studies have revealed that a glycoprotein fibulin-4 (FBLN4) is indispensable for the assembly of mature elastic fibers, it remains to be elucidated how FBLN4 takes part in elastogenesis. Here, we report a dose-dependent requirement for FBLN4 in the development of the elastic fibers in arteries, and a specific role of FBLN4 in recruiting the elastin-cross-linking enzyme, lysyl oxidase (LOX). Reduced expression of Fbln4, which was achieved with a smooth muscle-specific Cre-mediated gene deletion, caused arterial stiffness. Electron-microscopic examination revealed disorganized thick elastic laminae with aberrant deposition of elastin. Aneurysmal dilation of the ascending aorta was found when the Fbln4 expression level was reduced to an even lower level, whereas systemic Fbln4 null mice died perinatally from rupture of the diaphragm. We also found a specific interaction between FBLN4 and the propeptide of LOX, which efficiently promotes assembly of LOX onto tropoelastin. These data suggest a mechanism of elastogenesis, in which a sufficient amount of FBLN4 is essential for tethering LOX to tropoelastin to facilitate cross-linking. development | elastin | extracellular matrix www.pnas.org/cgi/doi/10.1073/pnas.0908268106

Muscular dystrophy is a general term encompassing muscle disorders that cause weakness and wasting, typically leading to premature death. Membrane instability, as a result of a genetic disruption within the dystrophin-glycoprotein complex (DGC), is thought to induce myofiber degeneration, although the downstream mechanism whereby membrane fragility leads to disease remains controversial. One potential mechanism that has yet to be definitively proven in vivo is that unregulated calcium influx initiates disease in dystrophic myofibers. Here we demonstrate that calcium itself is sufficient to cause a dystrophic phenotype in skeletal muscle independent of membrane fragility. For example, overexpression of transient receptor potential canonical 3 (TRPC3) and the associated increase in calcium influx resulted in a phenotype of muscular dystrophy nearly identical to that observed in DGC-lacking dystrophic disease models, including a highly similar molecular signature of gene expression changes. Furthermore, transgene-mediated inhibition of TRPC channels in mice dramatically reduced calcium influx and dystrophic disease manifestations associated with the mdx mutation (dystrophin gene) and deletion of the [delta]-sarcoglycan (Scgd) gene. These results demonstrate that calcium itself is sufficient to induce muscular dystrophy in vivo, and that TRPC channels are key disease initiators downstream of the unstable membrane that characterizes many types of muscular dystrophy. fibrosis | necrosis | degeneration | skeletal muscle www.pnas.org/cgi/doi/10.1073/pnas.0906591106

The proinflammatory cytokine TNF-[alpha] exerts its pleiotropic functions through activation of multiple downstream effectors, including JNK1. Yet, the underlying regulatory mechanism is incompletely understood. Here, we report that the transcription factor Myc-interacting zinc-finger protein 1 (Miz1) selectively suppresses TNF-[alpha]-induced JNK1 activation and cell death independently of its transcription activity. Proteomics analysis and yeast two-hybrid screening reveal that Mizl is a JNK-associated protein. The TNF-[alpha]induced activation of JNK1 is augmented in Miz1-deficient mouse embryonic fibroblasts ([Miz1.sup.-/-] MEFs), but the augmentation is abrogated by reintroduction of Miz1 or its transcription-deficient mutant. The regulation by Miz1 is highly specific, because it regulates TNF-[alpha]-induced TRAF2 K63-linked polyubiquitination. Neither JNK1 activation by IL-1[beta] or UV nor TNF-[alpha]-induced activation of p38, ERK, or I[kappa]B kinase complex is affected by the loss of Miz1. The TNF-[alpha]-induced cell death also is accelerated in [Miz1.sup.-/-] MEFs. Upon TNF-[alpha] stimulation, Miz1 is degraded rapidly by the proteasome, relieving its suppression on JNK1 activation. Thus, our results show that in addition to being a transcription factor Miz1 acts as a signal-and pathway-specific modulator or regulator that specifically regulates TNF-[alpha]-induced JNK1 activation and cell death. doi/10.1073/pnas.0906328106

The use of plasmon coupling in metal nanopartides has shown great potential for the optical characterization of many biological processes. Recently, we have demonstrated the use of 'plasmon rulers' to observe conformational changes of single biomolecules in vitro. Plasmon rulers provide robust signals without photo-bleaching or blinking. Here, we show the first application of plasmon rulers to in vivo studies to observe very long trajectories of single biomolecules in live cells. We present a unique type of plasmon ruler comprised of peptide-linked gold nanoparticle satellites around a core particle, which was used as a probe to optically follow cell-signaling pathways in vivo at the single-molecule level. These 'crown nanoparticle plasmon rulers' allowed us to continuously monitor trajectories of caspase-3 activity in live cells for over 2 h, providing sufficient time to observe early-stage caspase-3 activation, which was not possible by conventional ensemble analyses. caspase | live cell imaging | plasmonic nanoparticles | protease sensor | single-molecule imaging doi/ 10.1073/pnas.0907367106

In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules. electron cryomicroscopy | pracrystal | von Willebrand factor | tomography doi/ 10.1073/pnas.0902977106

Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting vessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, we uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor ceils from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development. 10.1126/science.1178577

Spermatogenesis can be divided into three stages: spermatogonial mitosis, meiosis of spermatocytes, and spermiogenesis. During spermiogenesis, spermatids undergo dramatic morphological changes including formation of a flagellum and chromosomal packaging and condensation of the nucleus into the sperm head. The genes regulating the latter processes are largely unknown. We previously discovered that a bi-functional gene, Spag16, is essential for spermatogenesis. SPAG16S, the 35 kDa, testis-specific isoform derived from the Spag16 gene, was found to bind to meiosis expressed gene 1 product (MEIG1), a protein originally thought to play a role in meiosis. We inactivated the Meigl gene and, unexpectedly, found that Meig1 mutant male mice had no obvious defect in meiosis, but were sterile as a result of impaired spermatogenesis at the stage of elongation and condensation. Transmission electron microscopy revealed that the manchette, a microtubular organelle essential for sperm head and flagellar formation was disrupted in spermatids of MEIG1-deficient mice. We also found that MEIG1 associates with the Parkin co-regulated gene (PACRG) protein, and that testicular PACRG protein is reduced in MEIGl-deficient mice. PACRG is thought to play a key role in assembly of the axonemes/flagella and the reproductive phenotype of Pacrg-deficient mice mirrors that of the Meigl mutant mice. Our findings reveal a critical role for the MEIG1/PARCG partnership in manchette structure and function and the control of spermiogenesis.

Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atgl protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway. cAMP-dependent protein kinase | macroautophagy | stationary phase | Tor protein kinase

High-throughput omics technologies generate huge datasets on the protein, transcript, lipid, and metabolite content of cells. By integrating and analyzing these data, systems biologists study complex networks of physical and functional interactions that go beyond the traditional focus on individual proteins or linear pathways. Many cell biologists have greeted these developments with healthy skepticism, complaining that long lists of genes or 'hairballs' of interactions provide little insight into biological questions of genuine meaning. As omics techniques move beyond acquisition into hypothesis-driven applications, the chasm between systems biologists and cell biologists is narrowing and the benefits of working together are increasingly clear. While cell biologists need omics and computer analyses to extend their understanding of biological processes, omics scientists need cell biologists to help them interpret and use their vast amounts of data.

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Forster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

Investigations into the biophysical properties of single molecules traditionally involve well defined in vitro systems where parameters such as solvent viscosity and applied forces are known a priori. These systems provide means to develop models describing the polymers response to a variety of conditions, including the entropically driven relaxation of a stretched biopolymer upon release of the tension inducing force. While these techniques have proven instrumental for recent advancements in the fields of polymer physics and biophysics, how applicable they are to life inside the cell remains poorly understood. Here we report an investigation of in vivo stretched polymer relaxation dynamics using chromatin relaxation following the breakage of a dicentric chromosome subjected to microtubule-based spindle forces. Additionally, we have developed an in vitro system used to verify the conformations observed during the in vivo relaxation, including the predicted but previously unidentified taut conformation. These observations motivate our use of existing polymer models to determine both the in vivo viscosity as seen by the relaxing chromatin and the tension force applied by the microtubule-based spindle in vivo. As a result, the technique described herein may be used as a biophysical strategy to probe the intranuclear environment. intranuclear viscosity | stem and flower

The use of an optical tweezer for moving dissociated neurons was studied. The main features of the tweezers are outlined as well as the general principles of its operation. Infrared beams at 980 and 1064 nm were used, focused so as to make a trap for holding neurons and moving them. Absorption by cells at those wavelengths is very small. Experiments were done to evaluate nonsticky substrate coatings, from which neurons could be easily lifted with the tweezers. The maximum speed of cell movement as a function of laser power was determined. Detailed studies of the damage to cells as a function of beam intensity and time of exposure were made. The 980 nm beam was much less destructive, for reasons that are not understood, and could be used to safely move cells through distances of millimeters in times of seconds. An illustrative application of the use of the tweezers to load neurons without damage into plastic cages on a glass substrate was presented. The conclusion is that optical tweezers are an accessible and practical tool for helping to establish neuron cultures of cells placed in specific locations. Index Terms--Cultured neurons, multielctrode array, optical tweezers.

Digital holographic microscopy enables a quantitative phase contrast metrology that is suitable for the investigation of reflective surfaces as well as for the marker-free analysis of living cells. The digital holographic feature of (subsequent) numerical focus adjustment makes possible applications for multifocus imaging. An overview of digital holographic microscopy methods is described. Applications of digital holographic microscopy are demonstrated by results obtained from livings cells and engineered surfaces. OCIS codes: 090.0090, 180.6900.

Thyroid pathology is a specialist area but is often encountered by the general pathologist in a variety of forms including cytology, frozen sections, and resection specimens. In the thyroid gland, [...]

Emerging technologies have enabled the acquisition of large genomics and proteomics data sets. However, current methodologies for analysis do not permit interpretation of the data in ways that unravel cellular networking. We propose a quantitative method for determining functional interactions in cellular signaling and gene networks. It can be used to explore cell systems at a mechanistic level or applied within a 'modular' framework, which dramatically decreases the number of variables to be assayed. This method is based on a mathematical derivation that demonstrates how the topology and strength of network connections can be retrieved from experimentally measured network responses to successive perturbations of all modules. Importantly, our analysis can reveal functional interactions even when the components of the system are not all known. Under these circumstances, some connections retrieved by the analysis will not be direct but correspond to the interaction routes through unidentified elements. The method is tested and illustrated by using computer-generated responses of a modeled mitogen-activated protein kinase cascade and gene network.

Research describes the use of a red fluorescent protein for subcellular localization studies in yeast cells. Data suggest that the red protein can be coupled to amino- and carboxy- terminals of suitable vectors.

An investigation of the mechanism involved in agonist-induced Ca2+ spikes using a caged inositol 1,4,5-trisphosphate (IP3) and BTC, a low-affinity Ca2+ indicator, in pancreatic acinar cells was conducted. The results, which included the rapid photolysis of the caged IP3 to reproduce acetylcholine-induced Ca2+ spikes, indicate the involvement of distinct mechanisms in local and global Ca2+ spikes. The kinetics of global Ca2+ spikes is concluded to be dependent on IP3 production.

In situ PCR amplification was used for the routine detection of specific bacterial cells at the single-cell level. The ECOL DNA primer set was used for the amplification of ribosomal DNA of Escherichia coli to identify cells at the single-cell level in the bacterial mixture. An epifluorescence microscope with in situ PCR produced high-contrast images. E coli cells in polluted river water were also observed using image analysis.

Experiments were conducted to determine how neocentromere activity, induced by genes of Abnormal chromosome 10 (Ab10), dominated the function of linked centromere/kinetochores in maize species. A four-dimensional system was utilized for the observation of the chromosome segregation in meiocytes. Results of the experiments revealed that neocentromeres were detected atthe metaphase-anaphase II stage. Disassembly of spindle fibers at kinetochores and forces generated by molecular motors at the centromere/kinetochore site were the two main reasons for the chromosome movement.