Annett Neuner

Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis

Cep164 provides a molecular link between the mother centriole and the ciliary membrane biogenesis machinery by interacting with the GEF Rabin8 and the GTPase Rab8.

Antiviral Activity and Mode of Action of Propolis Extracts and Selected Compounds

Aqueous and ethanol extracts of propolis were analysed phytochemically and examined for their antiviral activity in vitro. Different polyphenols, flavonoids and phenylcarboxylic acids were identified as major constituents. The antiviral effect of propolis extracts and selected constituents, e.g. caffeic acid (1), p‐coumaric acid (2), benzoic acid (3), galangin (4), pinocembrin (5) and chrysin (6) against herpes simplex virus type 1 (HSV‐1) was analysed in cell culture. The 50% inhibitory concentration (IC50) of aqueous and ethanol propolis extracts for HSV‐1 plaque formation was determined at 0.0004% and 0.000035%, respectively. Both propolis extracts exhibited high levels of antiviral activity against HSV‐1 in viral suspension tests, plaque formation was significantly reduced by >98%. In order to determine the mode of antiviral action of propolis, the extracts were added at different times during the viral infection cycle. Both propolis extracts exhibited high anti‐HSV‐1 activity when the viruses were pretreated with these drugs prior to infection. Among the analysed compounds, only galangin and chrysin displayed some antiviral activity. However, the extracts containing many different components exhibited significantly higher antiherpetic effects as well as higher selectivity indices than single isolated constituents. Propolis extracts might be suitable for topical application against herpes infection. Copyright

Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition

Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone‐assisted nuclear import via nuclear pores. The compartment‐specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter‐compartmental protein quality control system linked to DNA surveillance via INQ and Btn2.

Targeting of γ-tubulin complexes to microtubule organizing centers: conservation and divergence

Organisms with closed or open mitosis have differentially evolved various gamma-tubulin complex (γ-TuC) recruiting factors to organize diverse cellular microtubule (MT) arrays, including the mitotic spindle. γ-TuC recruiting factors not only target the γ-TuC to MT nucleation sites, but also regulate MT nucleation activity by generating the template for MT nucleation or promoting the MT nucleation activity of pre-existing γ-tubulin ring complexes (γ-TuRCs). Here we outline the current understanding of MT nucleator assembly and its regulation by γ-tubulin small complex (γ-TuSC) receptors. Moreover, we discuss the emergence of γ-TuC recruiting factors through evolution with augmented complexity and diversity and propose a hypothesis to account for the evolution of these factors in cooperative spindle assembly.

The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

A functional screen identified MARK4 as a positive regulator of axonemal extension and ciliogenesis via its interaction with the mother centriolar protein ODF2.

Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation

Budding yeast Spc110, a member of γ-tubulin complex receptor family (γ-TuCR), recruits γ-tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies suggest that Spc110 facilitates higher-order γ-tubulin complex assembly (Kollman et al., 2010). Nevertheless the molecular basis for this activity and the regulation are unclear. Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates γ-TuSC oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between the N-terminus of the γ-TuSC subunit Spc98 and Spc110 is important for this activity. Besides the conserved CM1 motif in γ-TuCRs (Sawin et al., 2004), a second motif that we named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/myomegalin) types of γ-TuCRs. The two types of γ-TuCRs contain distinct but conserved C-terminal MTOC targeting domains. DOI: http://dx.doi.org/10.7554/eLife.02208.001

Phosphorylation of the yeast γ-tubulin Tub4 regulates microtubule function.

The yeast γ-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in γ-tubulins from yeast to human.

An extended γ-tubulin ring functions as a stable platform in microtubule nucleation

Microtubule nucleation sites in yeast consist of a ring of γ-tubulin small complexes and a slight excess of uncomplexed γ-tubulin.

Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication

Spindle pole bodies (SPBs), like nuclear pore complexes, are embedded in the nuclear envelope (NE) at sites of fusion of the inner and outer nuclear membranes. A network of interacting proteins is required to insert a cytoplasmic SPB precursor into the NE. A central player of this network is Nbp1 that interacts with the conserved integral membrane protein Ndc1. Here, we establish that Nbp1 is a monotopic membrane protein that is essential for SPB insertion at the inner face of the NE. In vitro and in vivo studies identified an N‐terminal amphipathic α‐helix of Nbp1 as a membrane‐binding element, with crucial functions in SPB duplication. The karyopherin Kap123 binds to a nuclear localization sequence next to this amphipathic α‐helix and prevents unspecific tethering of Nbp1 to membranes. After transport into the nucleus, Nbp1 binds to the inner nuclear membrane. These data define the targeting pathway of a SPB component and suggest that the amphipathic α‐helix of Nbp1 is important for SPB insertion into the NE from within the nucleus.

MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template

Cells use γ-tubulin complex to nucleate microtubules. The assembly of active microtubule nucleator is spatially and temporally regulated through the cell cycle. Lin et al. show that the protein Mzt1/MOZART1 and γ-tubulin complex receptors directly interact and act together to assemble the γ-tubulin small complex into an active microtubule nucleation template and that such interaction is conserved between Candida albicans and human cells.