Nonmuscle myosin-2: mix and match

Members of the nonmuscle myosin-2 (NM-2) family of actin-based molecular motors catalyze the conversion of chemical energy into directed movement and force thereby acting as central regulatory components of the eukaryotic cytoskeleton. By cyclically interacting with adenosine triphosphate and F-actin, NM-2 isoforms promote cytoskeletal force generation in established cellular processes like cell migration, shape changes, adhesion dynamics, endo- and exo-cytosis, and cytokinesis. Novel functions of the NM-2 family members in autophagy and viral infection are emerging, making NM-2 isoforms regulators of nearly all cellular processes that require the spatiotemporal organization of cytoskeletal scaffolding. Here, we assess current views about the role of NM-2 isoforms in these activities including the tight regulation of NM-2 assembly and activation through phosphorylation and how NM-2-mediated changes in cytoskeletal dynamics and mechanics affect cell physiological functions in health and disease

Umweltmanagement und ECO-Design: Dokumentation der transnationalen Partnerschaft zwischen Verein Faktor 4+, Klagenfurt, Wuppertal Institut, Wuppertal, Klaus Novy Institut, Köln. Zukunftsfähige Unternehmen (4)

--Ressourcenmanagement,kleine und mittlere Unternehmen,Beschäftigung,Qualifizierung und Beteiligung,Eco-design,MIPS,Öko-Effizienz,Faktor 4/10,EG-Öko-Audit-Verordnung,Umweltmanagement,Resource management,small and medium sized companies,employment,qualification,participation,Eco-design,MIPS,eco-efficiency,factor 4/10,EMAS,environmental management

Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion.

MYO1C, a single-headed class I myosin, associates with cholesterol-enriched lipid rafts and facilitates their recycling from intracellular compartments to the cell surface. Absence of functional MYO1C disturbs the cellular distribution of lipid rafts, causes the accumulation of cholesterol-enriched membranes in the perinuclear recycling compartment, and leads to enlargement of endolysosomal membranes. Several feeder pathways, including classical endocytosis but also the autophagy pathway, maintain the health of the cell by selective degradation of cargo through fusion with the lysosome. Here we show that loss of functional MYO1C leads to an increase in total cellular cholesterol and its disrupted subcellular distribution. We observe an accumulation of autophagic structures caused by a block in fusion with the lysosome and a defect in autophagic cargo degradation. Interestingly, the loss of MYO1C has no effect on degradation of endocytic cargo such as EGFR, illustrating that although the endolysosomal compartment is enlarged in size, it is functional, contains active hydrolases, and the correct pH. Our results highlight the importance of correct lipid composition in autophagosomes and lysosomes to enable them to fuse. Ablating MYO1C function causes abnormal cholesterol distribution, which has a major selective impact on the autophagy pathway.This work was financially supported by the Wellcome Trust (F.B., D.A.T. and H.B.), the Deutsche Forschungsgemeinschaft Grant MA 1081/19–1 (D.J.M) and the Medical Research Council (F.B and C. K.-I.). The CIMR is in receipt of a strategic award from the Wellcome Trust (100140).This is the final published version. It first appeared at http://www.tandfonline.com/doi/abs/10.4161/15548627.2014.984272#.VNo0Gy6Qne4

Structural and biochemical studies of sulphotransferase 18 from Arabidopsis thaliana explain its substrate specificity and reaction mechanism

Sulphotransferases are a diverse group of enzymes catalysing the transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulphate (PAPS) to a broad range of secondary metabolites. They exist in all kingdoms of life. In Arabidopsis thaliana (L.) Heynh. twenty-two sulphotransferase (SOT) isoforms were identified. Three of those are involved in glucosinolate (Gl) biosynthesis, glycosylated sulphur-containing aldoximes containing chemically different side chains, whose break-down products are involved in stress response against herbivores, pathogens, and abiotic stress. To explain the differences in substrate specificity of desulpho (ds)-Gl SOTs and to understand the reaction mechanism of plant SOTs, we determined the first high-resolution crystal structure of the plant ds-Gl SOT AtSOT18 in complex with 3'-phosphoadenosine 5'-phosphate (PAP) alone and together with the Gl sinigrin. These new structural insights into the determination of substrate specificity were complemented by mutagenesis studies. The structure of AtSOT18 invigorates the similarity between plant and mammalian sulphotransferases, which illustrates the evolutionary conservation of this multifunctional enzyme family. We identified the essential residues for substrate binding and catalysis and demonstrated that the catalytic mechanism is conserved between human and plant enzymes. Our study indicates that the loop-gating mechanism is likely to be a source of the substrate specificity in plants.DFG/PA 764/10-1DFG/FE 1510/2-1EC/Marie Curie Fellowship 625451 SUPA-H

Contact-controlled amoeboid motility induces dynamic cell trapping in 3D-microstructured surfaces.

On flat substrates, several cell types exhibit amoeboid migration, which is characterized by restless stochastic successions of pseudopod protrusions. The orientation and frequency of new membrane protrusions characterize efficient search modes, which can respond to external chemical stimuli as observed during chemotaxis in amoebae. To quantify the influence of mechanical stimuli induced by surface topography on the migration modes of the amoeboid model organism Dictyostelium discoideum, we apply high resolution motion analysis in microfabricated pillar arrays of defined density and geometry. Cell motion is analyzed by a two-state motility-model, distinguishing directed cellular runs from phases of isotropic migration that are characterized by randomly oriented cellular protrusions. Cells lacking myosin II or cells deprived of microtubules show significantly different behavior concerning migration velocities and migrational angle distribution, without pronounced attraction to pillars. We conclude that microtubules enhance cellular ability to react with external 3D structures. Our experiments on wild-type cells show that the switching from randomly formed pseudopods to a stabilized leading pseudopod is triggered by contact with surface structures. These alternating processes guide cells according to the available surface in their 3D environment, which we observed dynamically and in steady-state situations. As a consequence, cells perform "home-runs" in low-density pillar arrays, crawling from pillar to pillar, with a characteristic dwell time of 75 s. At the boundary between a flat surface and a 3D structured substrate, cells preferentially localize in contact with micropillars, due to the additionally available surface in the microstructured arrays. Such responses of cell motility to microstructures might open new possibilities for cell sorting in surface structured arrays

Shedding light on the variability of optical skin properties: finding a path towards more accurate prediction of light propagation in human cutaneous compartments

YesFinding a path towards a more accurate prediction of light propagation in human skin remains an aspiration of biomedical scientists working on cutaneous applications both for diagnostic and therapeutic reasons. The objective of this study was to investigate variability of the optical properties of human skin compartments reported in literature, to explore the underlying rational of this variability and to propose a dataset of values, to better represent an in vivo case and recommend a solution towards a more accurate prediction of light propagation through cutaneous compartments. To achieve this, we undertook a novel, logical yet simple approach. We first reviewed scientific articles published between 1981 and 2013 that reported on skin optical properties, to reveal the spread in the reported quantitative values. We found variations of up to 100-fold. Then we extracted the most trust-worthy datasets guided by a rule that the spectral properties should reflect the specific biochemical composition of each of the skin layers. This resulted in the narrowing of the spread in the calculated photon densities to 6-fold. We conclude with a recommendation to use the identified most robust datasets when estimating light propagation in human skin using Monte Carlo simulations. Alternatively, otherwise follow our proposed strategy to screen any new datasets to determine their biological relevance.European Marie-Curie Actions Programme, Grant agreement no. 60788

Process control and in silico modeling strategies for enabling high density culture of human pluripotent stem cells in stirred tank bioreactors

The routine therapeutic and industrial applications of human pluripotent stem cells (hPSCs) require their constant mass supply by robust, efficient, and economically viable bioprocesses. Our protocol describes the fully controlled expansion of hPSCs in stirred tank bioreactors (STBRs) enabling cell densities of 35 3 106 cells/mL while reducing culture medium consumption by 75%. This is achieved by in silico process modeling and computable upscaling. We provide a detailed blueprint for systematic process development of hPSCs and their progenies

3D structure of Thermus aquaticus single-stranded DNA–binding protein gives insight into the functioning of SSB proteins

In contrast to the majority of tetrameric SSB proteins, the recently discovered SSB proteins from the Thermus/Deinoccus group form dimers. We solved the crystal structures of the SSB protein from Thermus aquaticus (TaqSSB) and a deletion mutant of the protein and show the structure of their ssDNA binding domains to be similar to the structure of tetrameric SSBs. Two conformations accompanied by proline cis–trans isomerization are observed in the flexible C-terminal region. For the first time, we were able to trace 6 out of 10 amino acids at the C-terminus of an SSB protein. This highly conserved region is essential for interaction with other proteins and we show it to adopt an extended conformation devoid of secondary structure. A model for binding this region to the χ subunit of DNA polymerase III is proposed. It explains at a molecular level the reason for the ssb113 phenotype observed in Escherichia coli

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin-Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy-Causing Double Mutation MYBPC3Δ25bp/D389V

Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein–protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0–C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages