Function and organisation of actin and septins in Neurospora crassa

This thesis deals with the organisation and function of actin and septins in the model filamentous fungus, Neurospora crassa. Firstly, study demonstrates the utility of the Lifeact peptide probe for the investigation of actin dynamics in N. crassa. Lifeact fused to fluorescent proteins allowed live-cell imaging of actin patches, cables and rings without interfering with cellular functions. Actin cables and patches localised to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organisation was markedly different between the tip regions of mature hyphae and germ tubes. Only mature hyphae displayed a sub-apical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Proper organisation of actin cables required the class-V myosin, MYO-5, and the frequency of rapid transport of actin patches was reduced in its absence, suggesting that MYO-5 participates in actin patch translocation. Deletion of myo-5 caused gross morphological and polarity defects, demonstrating the importance of this motor for normal cell function. GFP-tagged MYO-5 localised as a crescent at germ tube tips and to the core of the Spitzenkörper in mature hyphae. Secondly, analysis of septin null mutants demonstrated that septins limit the emergence of germ tubes and are important for septation and conidiation in N. crassa. Septins showed different patterns of localisation at hyphal tips, with GFP-CDC-10 and CDC- 11-GFP organised as a collar with lower signal intensity at the tip apex, CDC-3-GFP and CDC-12-GFP constituted as a cap at the tip apex and GFP-SPN-1 forming an extended collar. Septins formed a range of different higher-order structures in N. crassa – rings, loops, fibres, bar-like structures, and caps – which can co-exist within the same cell. Purification of the septin complex and mass spectrometry of isolated proteins revealed that the septin complex consists predominantly of CDC-3, CDC-10, CDC-11 and CDC-12. Immunoprecipitation of SPN-1 revealed that this septin interacts with the core septin complex

Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink

Increasing the efficiency of the conversion of light energy to products by photosynthesis represents a grand challenge in biotechnology. Photosynthesis is limited by the carbon-fixing enzyme Rubisco resulting in much of the absorbed energy being wasted as heat or fluorescence or lost as excess reductant via alternative electron dissipation pathways. To harness this wasted reductant, we engineered the model cyanobacterium Synechococcus PCC 7002 to express the mammalian cytochrome P450 CYP1A1 to serve as an artificial electron sink for excess electrons derived from light-catalyzed water-splitting. This improved photosynthetic efficiency by increasing the maximum rate of photosynthetic electron flow by 31.3%. A simple fluorescent assay for CYP1A1 activity demonstrated that the P450 was functional in the absence of its native reductase, that activity was light-dependent and scaled with irradiance. We show for the first time in live cells that photosynthetic reductant can be redirected to power a heterologous cytochrome P450. Furthermore, Synechococcus PCC 7002 expressing CYP1A1 degraded the herbicide atrazine, which is a widespread environmental pollutant

Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes.

This is the final version of the article. Available from Rockefeller University Press via the DOI in this record.Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood. In this paper, we report that a Hook protein, Hok1, is essential for kinesin-3- and dynein-dependent early endosome (EE) motility in the fungus Ustilago maydis. Hok1 binds to EEs via its C-terminal region, where it forms a complex with homologues of human fused toes (FTS) and its interactor FTS- and Hook-interacting protein. A highly conserved N-terminal region is required to bind dynein and kinesin-3 to EEs. To change the direction of EE transport, kinesin-3 is released from organelles, and dynein binds subsequently. A chimaera of human Hook3 and Hok1 rescues the hok1 mutant phenotype, suggesting functional conservation between humans and fungi. We conclude that Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional EE trafficking by controlling attachment of both kinesin-3 and dynein.This work was supported by Wellcome Trust (097835/Z/11/Z) and the Biotechnology and Biological Sciences Research Council (BB/J009903/1)

F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth

Ru(II)-diimine complexes and cytochrome P450 working hand-in-hand

With a growing interest in utilizing visible light to drive biocatalytic processes, several light-harvesting units and approaches have been employed to harness the synthetic potential of heme monooxygenases and carry out selective oxyfunctionalization of a wide range of substrates. While the fields of cytochrome P450 and Ru(II) photochemistry have separately been prolific, it is not until the turn of the 21st century that they converged. Non-covalent and subsequently covalently attached Ru(II) complexes were used to promote rapid intramolecular electron transfer in bacterial P450 enzymes. Photocatalytic activity with Ru(II)-modified P450 enzymes was achieved under reductive conditions with a judicious choice of a sacrificial electron donor. The initial concept of Ru(II)-modified P450 enzymes was further improved using protein engineering, photosensitizer functionalization and was successfully applied to other P450 enzymes. In this review, we wish to present the recent contributions from our group and others in utilizing Ru(II) complexes coupled with P450 enzymes in the broad context of photobiocatalysis, protein assemblies and chemoenzymatic reactions. The merging of chemical catalysts with the synthetic potential of P450 enzymes has led to the development of several chemoenzymatic approaches. Moreover, strained Ru(II) compounds have been shown to selectively inhibit P450 enzymes by releasing aromatic heterocycle containing molecules upon visible light excitation taking advantage of the rapid ligand loss feature in those complexes

Myosin concentration underlies cell size–dependent scalability of actomyosin ring constriction

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution 3.0 License. The definitive version was published in Journal of Cell Biology 195 (2011): 799-813, doi:10.1083/jcb.201101055.In eukaryotes, cytokinesis is accomplished by an actomyosin-based contractile ring. Although in Caenorhabditis elegans embryos larger cells divide at a faster rate than smaller cells, it remains unknown whether a similar mode of scalability operates in other cells. We investigated cytokinesis in the filamentous fungus Neurospora crassa, which exhibits a wide range of hyphal circumferences. We found that N. crassa cells divide using an actomyosin ring and larger rings constricted faster than smaller rings. However, unlike in C. elegans, the total amount of myosin remained constant throughout constriction, and there was a size-dependent increase in the starting concentration of myosin in the ring. We predict that the increased number of ring-associated myosin motors in larger rings leads to the increased constriction rate. Accordingly, reduction or inhibition of ring-associated myosin slows down the rate of constriction. Because the mechanical characteristics of contractile rings are conserved, we predict that these findings will be relevant to actomyosin ring constriction in other cell types.Work in the laboratories of M.K. Balasubramanian and G. Jedd is supported by research funds from Singapore Millennium Foundation and the Temasek Life Sciences Laboratory.2012-05-2

CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa

Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms. We demonstrate that RAC-1 is essential for CAT formation and cell fusion, whereas CDC-42 is necessary and sufficient for normal germ tube development. Cdc42-Rac-interactive-binding (CRIB) reporters were constructed to exclusively label locally activated GTP-bound GTPases. Time course analyses showed that repositioning of these activated GTPase clusters within germ tube and CAT tip apices controls directional growth in the absence of a tip-localized vesicle supply center (Spitzenkörper). We propose a model in which the local assembly of a plasma-membrane-associated GTPase-PAK-MAPK signaling platform regulates chemoattractant perception and secretion in order to synchronize oscillatory cell-cell communication and directional CAT tip growth

Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome

A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture