A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles

Imaging datasets are rich in quantitative information. However, few cell biologists possess the tools necessary to analyze them. Here, we present a large dataset of Xenopus extract spindle images together with an analysis pipeline designed to assess spindle morphology across a range of experimental conditions. Our analysis of different spindle types illustrates how kinetochore microtubules amplify spindle microtubule density. Extract mixing experiments reveal that some spindle features titrate, while others undergo switch-like transitions, and multivariate analysis shows the pleiotropic morphological effects of modulating the levels of TPX2, a key spindle assembly factor. We also apply our pipeline to analyze nuclear morphology in human cell culture, showing the general utility of the segmentation approach. Our analyses provide new insight into the diversity of spindle types and suggest areas for future study. The approaches outlined can be applied by other researchers studying spindle morphology and adapted with minimal modification to other experimental systems.     

Avoiding a crisis of motivation for ocean management under global environmental change

Climate change and ocean acidification are altering marine ecosystems and, from a human perspective, creating both winners and losers. Human responses to these changes are complex, but may result in reduced government investments in regulation, resource management, monitoring and enforcement. Moreover, a lack of peoples’ experience of climate change may drive some towards attributing the symptoms of climate change to more familiar causes such as management failure. Taken together, we anticipate that management could become weaker and less effective as climate change continues. Using diverse case studies, including the decline of coral reefs, coastal defences from flooding, shifting fish stocks and the emergence of new shipping opportunities in the Arctic, we argue that human interests are better served by increased investments in resource management. But greater government investment in management does not simply mean more of “business‐as‐usual.” Management needs to become more flexible, better at anticipating and responding to surprise, and able to facilitate change where it is desirable. A range of technological, economic, communication and governance solutions exists to help transform management. While not all have been tested, judicious application of the most appropriate solutions should help humanity adapt to novel circumstances and seek opportunity where possible.     

The Hepatitis C virus NS5A protein activates a phosphoinositide 3-kinase-dependent survival signaling cascade

Hepatitis C virus (HCV) establishes a persistent infection, with up to 80% of infected individuals proceeding to chronic hepatitis, which in many cases may result in liver cirrhosis and hepatocellular carcinoma (HCC); indeed HCV infection is increasingly associated with the development of HCC. The long time period (up to 30 years) between primary infection and the onset of HCC implies that HCV is not directly oncogenic but in some way predisposes patients to develop tumors, though the mechanism for this is unclear as yet. We report here that NS5A binds directly to the Src homology 3 domain of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K), and this interaction is mediated by a novel (non-proline-rich) motif within NS5A. Coimmunoprecipitation analysis revealed that NS5A bound native heterodimeric PI3K and enhanced the phosphotransferase activity of the catalytic (p110) subunit both in vitro and in human cell lines harboring a subgenomic HCV replicon or expressing NS5A alone. NS5A-mediated activation of PI3K resulted in increased phosphorylation and activity of Akt/protein kinase B and concomitantly provided protection against the induction of apoptosis in both replicon-harboring cells and cells stably expressing NS5A alone. These data suggest that stimulation of PI3K by NS5A may represent an indirect mechanism for development of HCC in HCV-infected patients and further suggests potential therapeutic strategies to counteract the occurrence of HCV-related HCC.     

1H NMR and NOE studies of the purple acid phosphatases from porcine uterus and bovine spleen.

The diiron active sites of the purple acid phosphatases from porcine uterus (also called uteroferrin, Uf) and bovine spleen (BSPAP) and their complexes with tungstate are compared by 1H NMR and NOE techniques. The paramagnetically shifted features of the 1H NMR spectrum of reduced BSPAP are similar to those of reduced Uf, while the spectra of the tungstate complexes are almost identical. These observations suggest that the two active sites are quite similar, in agreement with the greater than 90% sequence homology found in the two enzymes. Nuclear Overhauser effect (NOE) experiments on the His N-H resonances show that the Fe(III)-His residue is N epsilon-coordinated, while the Fe(II)-His is H delta-coordinated in both enzymes. On the basis of the above NMR and NOE results, our previously proposed model for the dinuclear iron active site of Uf [Scarrow, R. C., Pyrz, J. W., & Que, L., Jr. (1990) J. Am. Chem. Soc. 112, 657-665] is corroborated, refined, and found to represent the diiron center of BSPAP as well.     

Metal contamination in sediments and biota of the Bay of Quinte,Lake Ontario,Canada

The Bay of Quinte receives drainage from several large river systems, including the Moira River which carried sediment from mines into the Bay from the 1880s to the 1960s. We are investigating possible metal contamination of submerged weed beds and marsh biota which may contribute to the low diversity and biomass of macrophyte beds and Typha marshes in the Bay. In 1987, sediment, macrophytes and snails were sampled in wetlands close to the Moira River and at Hay Bay (part of the Bay of Quinte presumably unaffected by mine effluents) located 20 km from the Moira. Some element concentrations in sediment and biota were determined by neutron activation analysis (NAA) including Al, As, Br, Ca, Co, Cl, Cr, Cs, Fe, Hf, K, La, Na, Mg, Sb, Sc, Rb, Ta, Th, Ti, U, V and Zn. Other elements were analysed by acid dissolution and atomic absorption spectrophotometry (AAS) including Ag, As, Cu, Hg, Ni, Pb, and Zn. Levels of As in sediments and plants were higher close to the Moira River, whereas Cu and Ni showed the opposite pattern in sediments. The usefulness of species as bioassays differed: Stagnicola elodes Say accumulated significantly higher levels of Cu (35 vs 18 ppm) and V (l. l vs 0.5 ppm) than Planorbella trivolvis Say collected from the same sites. The macrophyte, Myriophyllum spicatum L. acted as an accumulator of Pb (up to 9.6 ppm), whereas Pb in Vallisneria americana Michx. at the same sites was undetectable.     

Mutational Analysis of the Transmembrane Helix 2-HAMP Domain Connection in the Escherichia coli Aspartate Chemoreceptor Tar

Transmembrane helix 2 (TM2) of the Tar chemoreceptor undergoes an inward piston-like displacement of 1 to 3 Å upon binding aspartate. This signal is transmitted to the kinase-control module via the HAMP domain. Within Tar, the HAMP domain forms a parallel four-helix bundle consisting of a dimer of two amphipathic helices connected by a flexible linker. In the nuclear magnetic resonance structure of an archaeal HAMP domain, residues corresponding to the MLLT sequence between Arg-214 at the end of TM2 and Pro-219 of Tar are an N-terminal helical extension of AS1. We modified this region to test whether it behaves as a continuous helical connection between TM2 and HAMP. First, one to four Gly residues were inserted between Thr-218 and Pro-219. Second, the MLLT sequence was replaced with one to nine Gly residues. Third, the sequence was shortened or extended with residues compatible with helix formation. Cells expressing receptors in which the MLLT sequence was shortened to MLL or in which the MLLT sequence was replaced by four Gly residues performed good aspartate chemotaxis. Other mutant receptors supported diminished aspartate taxis. Most mutant receptors had biased signal outputs and/or abnormal patterns of adaptive methylation. We interpret these results to indicate that a strong, permanent helical connection between TM2 and the HAMP domain is not necessary for normal transmembrane signaling.The HAMP domain is a structural motif commonly found in histidine kinases (HKs), adenylate cyclases, methyl-accepting chemotaxis proteins (MCPs), and phosphatases (2). In Escherichia coli and Salmonella enterica MCPs, the HAMP domain is located between a transmembrane-sensing module composed of ligand-binding and transmembrane regions and a kinase-control module composed of adaptation and kinase-activating regions (Fig. ​(Fig.1A)1A) (19). Therefore, HAMP domains are responsible for bidirectional signal transduction between these modules.Open in a separate windowFIG. 1.Domain architecture of the aspartate chemoreceptor. (A) The cartoon, based on a figure from Hazelbauer et al. (2008) (19), illustrates the architecture of the aspartate chemoreceptor. Protein structural domains are labeled on the left, and functional modules are labeled on the right. (B) Schematic of TM2 and the control cable region attached to a ribbon diagram of the solution NMR structure of the Af1503 HAMP domain four-helix bundle (22). TM2 is shown in purple within the membrane. The control cable of Tar_Ec consists of 5 amino acyl residues (Gly-Ile-Arg-Arg-Met) that connect TM2 and AS1 of HAMP. AS1 is shown in blue, AS2 is shown in red, and the 14-residue AS1-AS2 connector (CTR) is shown in black. The residue equivalent to Arg-214 in Tar_Ec is also highlighted in blue, the conserved Pro residue (Pro-219 in Tar_Ec) is highlighted in yellow, and residues equivalent to the MLLT sequence between TM2 and Pro-219 in Tar_Ec are highlighted in cyan.The determination of a high-resolution three-dimensional structure of a HAMP domain from an MCP remains elusive. However, a solution nuclear magnetic resonance (NMR) structure of a HAMP domain has been determined for the Af1503 protein of unknown function from the archaeal thermophile Archeoglobus fulgidus (22). The domain forms a parallel four-helix bundle, with two amphipathic helices, AS1 and AS2, being contributed by each subunit (Fig. ​(Fig.1B).1B). In this structure, the helices pack in an unusual x-da configuration, commonly referred to as knobs-to-knobs packing, in which the large hydrophobic x residues stabilize both intrasubunit and intersubunit interactions.Evidence for the existence of a four-helix HAMP bundle within intact receptors comes from disulfide cross-linking experiments with Salmonella enterica Tar (Tar_Se) (41) and the E. coli Aer redox receptor (Aer_Ec) (44). In vivo genetic studies (1, 48) are also consistent with the existence of a four-helix bundle in the E. coli Tsr receptor (Tsr_Ec).Tar_Ec functions as the aspartate chemoreceptor in E. coli (39). Each monomer within the homodimeric (15, 29) receptor possess a periplasmic ligand-binding domain composed of four antiparallel α helices that form four-helix bundles (8). The transmembrane regions that flank this periplasmic domain (transmembrane helix 1 [TM1] and TM2) are extensions of the periplasmic helices PD1 and PD4 (27, 33, 37, 40). Aspartate binds at either of two rotationally symmetrical sites at the dimer interface. Each binding site contains residues from PD1 of one subunit and PD1′ and PD4′ of the other. Aspartate binding generates a small (∼1- to 3-Å) vertical displacement into the cytoplasm of one contiguous PD4-TM2 helix relative to the other (14).E. coli Tar (Tar_Ec) and other chemoreceptors normally activate the histidine protein kinase CheA (5), which is coupled to the receptors via the adapter protein CheW. CheA autophosphorylates, and the phosphoryl group is subsequently transferred to the response regulator CheY (21). CheY-P interacts with FliM within the flagellar motor to promote clockwise (CW) rotation of the flagella (36, 46). Counterclockwise (CCW) motor rotation allows the flagellar filaments to coalesce into a bundle that propels the cell in a run (38). CW rotation of one or more flagella disrupts the bundle and generates a tumble (43). Therefore, the relative activities of CheA and the CheY-P phosphatase, CheZ, establish the ratio of CheY to CheY-P within the cell and hence the frequency of tumbling (11, 21).The conformational changes induced by aspartate convert Tar_Ec from a stimulator of CheA activity into an inhibitor (6). The resulting drop in CheY-P activity, which is accelerated by CheZ, suppresses tumbling and lengthens the average run. Inhibition of CheA activity is reversed by covalent methylation of the cognate receptor (17). Methylation is facilitated by a transient decrease in the level of the active, phosphorylated form of the CheB methylesterase (26), which is another substrate for phosphotransfer from CheA (21). The well-studied properties of this system, and the possibility of monitoring several different in vivo parameters, make it amenable for examining signal transduction between TM2 and the adjoining HAMP domain.The apical region of AS1 in Tar_Ec is composed of the tetrapeptide Met-Leu-Leu-Thr, which connects residue Arg-214 at the end of TM2 with the conserved Pro-219 residue within AS1 of the HAMP domain (Fig. ​(Fig.1B).1B). The corresponding sequence is Thr-Ile-Thr-Arg in the Af1503 HAMP domain. In the NMR structure of the isolated Af1503 HAMP, the corresponding four residues (Thr-Ile-Thr-Arg) comprise an unpaired helical extension of the N terminus of AS1 before Pro-283 (9, 22), but it is unclear how these residues may pack in an intact membrane-spanning protein. In the Af1503 HAMP, Pro-283 packs against residues Glu-311 and Ile-312, which form the N terminus of AS2. The residues at the equivalent positions in Tar_Ec are Glu-246 and Met-247 (Fig. ​(Fig.1B1B).We modified the length and residue composition of the region between Arg-214 and Pro-219 in Tar_Ec in different ways and monitored the ability of the mutant proteins to support chemotactic migration, to generate the clockwise flagellar rotation that reflects CheA activation, and to regulate adaptive methylation. The results support a model in which the structural tension exerted by TM2 on AS1 controls its signaling state, as proposed by Zhou et al. (48). In the context of that model, the results suggest that when the receptor is in the kinase-inhibiting state, the HAMP domain is in a stable four-helix bundle.     

Ecological and morphological shifts in Lake Michigan fishes: glimpses of the ghost of competition past

Synopsis Both historical patterns and recent evidence of resource partitioning and complementarity within the Lake Michigan fish community provide circumstantial evidence for interspecific competition. But competition is difficult to document in the field without controlled experimentation. In Lake Michigan, controlled experiments on competition within the fish community are nearly impossible, but we still need to understand the interactions among the dominant fishes. For this purpose, I have relied upon hypothesis-based field observation, natural experiments in the field and designed laboratory experiments to evaluate competitive interactions. Resource use patterns and trophic morphology of the bloater, Coregonus hoyi, a native cisco, from samples taken before alewife, Alosa pseudoharengus, became abundant (1960) were compared to more recent data (1979–80). After the alewife density increase, bloaters had significantly fewer and shorter gill rakers. This suggests a morphological shift toward greater benthic foraging efficiency in response to high abundances of an efficient pelagic planktivore, alewife. Resource use comparisons suggested that bloaters now shift from pelagic zooplanktivory to benthic habitats and diets at least two years earlier in their life history than they did before alewife became abundant. This evidence, albeit not experimental, provides strong support for the importance of competition in the structure of the current Lake Michigan fish community.In Lake Michigan, seasonal thermal habitat compression can pack fish into a narrow thermal zone across the lake bottom, leading to increased habitat overlap, reduced prey availability and fish diets containing fewer and smaller prey. Thermal habitat compression, which can occur intermittently through the season, may create competitive bottlenecks which help maintain the observed resource partitioning among these fishes.     

A conditional allele of Rspo3 reveals redundant function of R‐spondins during mouse limb development

Summary: R‐spondins are secreted ligands that bind cell surface receptors and activate Wnt/β‐catenin signaling. Human mutations and gene inactivation studies in mice have revealed a role for these four proteins (RSPO1‐4) in diverse developmental processes ranging from sex determination to limb development. Among the genes coding for R‐spondins, only inactivation of Rspo3 shows early embryonic lethality (E10.5 in mice). Therefore, a conditional allele of this gene is necessary to understand the function of R‐spondins throughout murine development. To address this need, we have produced an allele in which loxP sites flank exons 2–4 of Rspo3, allowing tissue‐specific deletion of these exons in the presence of Cre recombinase. We used these mice to investigate the role of Rspo3 during limb development and found that limbs ultimately developed normally in the absence of Rspo3 function. However, severe hindlimb truncations resulted when Rspo3 and Rspo2 mutations were combined, demonstrating redundant function of these genes. genesis 50:741–749, 2012. © 2012 Wiley Periodicals, Inc.