Functional analysis of phosphorylation of the mitotic centromere-associated kinesin by Aurora B kinase in human tumor cells

Mitotic centromere-associated kinesin (MCAK) is the best characterized member of the kinesin-13 family and plays important roles in microtubule dynamics during mitosis. Its activity and subcellular localization is tightly regulated by an orchestra of mitotic kinases, such as Aurora B. It is well known that serine 196 of MCAK is the major phosphorylation site of Aurora B in Xenopus leavis extracts and that this phosphorylation regulates its catalytic activity and subcellular localization. In the current study, we have addressed the conserved phosphorylation site serine 192 in human MCAK to characterize its function in more depth in human cancer cells. Our data confirm that S192 is the major phosphorylation site of Aurora B in human MCAK and that this phosphorylation has crucial roles in regulating its catalytic activity and localization at the kinetochore/centromere region in mitosis. Interfering with this phosphorylation leads to a delayed progression through prometa- and metaphase associated with mitotic defects in chromosome alignment and segregation. We show further that MCAK is involved in directional migration and invasion of tumor cells, and interestingly, interference with the S192 phosphorylation affects this capability of MCAK. These data provide the first molecular explanation for clinical observation, where an overexpression of MCAK was associated with lymphatic invasion and lymph node metastasis in gastric and colorectal cancer patients.     

Navigating the plant cell: intracellular transport logistics in the green kingdom

Intracellular transport in plant cells occurs on microtubular and actin arrays. Cytoplasmic streaming, the rapid motion of plant cell organelles, is mostly driven by an actin–myosin mechanism, whereas specialized functions, such as the transport of large cargo or the assembly of a new cell wall during cell division, are performed by the microtubules. Different modes of transport are used, fast and slow, to either haul cargo over long distances or ascertain high-precision targeting, respectively. Various forms of the actin-specific motor protein myosin XI exist in plant cells and might be involved in different cellular functions.     

Coiled-Coil Formation on Lipid Bilayers—Implications for Docking and?Fusion Efficiency

Coiled-coil formation of four different oligopeptides was characterized in solution, on hydrogels, and on membranes by employing circular dichroism spectroscopy, surface plasmon resonance spectroscopy, attenuated total reflection infrared spectroscopy, and ellipsometry. Peptide sequences rich in either glutamic acid (E: E3Cys, i-E3Cys) or lysine (K: K3Cys, i-K3Cys) were used to represent minimal mimics of eukaryotic SNARE motifs. Half of the peptides were synthesized in reverse sequence, so that parallel and antiparallel heptad coiled-coil structures were formed. Either E-peptides or K-peptides were attached covalently to phospholipid anchors via maleimide chemistry, and served as receptors for the recognition of the corresponding binding partners added to solution. Attenuated total reflection infrared spectroscopy of single bilayers confirmed the formation of coiled-coil complexes at the membrane interface. Coiled-coil formation in solution, as compared with association at the membrane surface, displays considerably larger binding constants that are largely attributed to loss of translational entropy at the interface. Finally, the fusogenicity of the various coiled-coil motifs was explored, and the results provide clear evidence that hemifusion followed by full fusion requires a parallel orientation of α-helices, whereas antiparallel oriented coiled-coil motifs display only docking.     

Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles of the motif for the voltage-dependent and voltage-independent gating, we performed reciprocal replacements of the asparagine and aspartate within the S3 motif in both channels, following the proposed idea that specific electrostatic interactions with other domains take place during gating. Wild-type and mutant channels were heterologeously expressed in HEK-293 cells and channel function was analyzed by whole-cell patch-clamp analysis as well as by Ca²⁺-imaging. Additionally, the expression of the channels in the plasma membrane was tested by Western blot analysis, in part after biotinylation. For the mutations of TRPM8, responses to menthol were only compromised if also the expression of the glycosylated channel isoform was prevented. In contrast, responses to cold were consistently and significantly attenuated but not completely abolished. For TRPM2, surface expression was not significantly affected by any of the mutations but channel function was only retained in one variant. Remarkably, this was the variant of which the corresponding mutation in TRPM8 exerted the most negative effects both on channel function and expression. Furthermore, we performed an exchange of the inner pair of residues of the N-x-x-D motif between the two channels, which proved deleterious for the functional expression of TRPM8 but ineffective on TRPM2. In conclusion, the N-x-x-D motif plays specific roles in TRPM8 and TRPM2, reflecting different requirements for voltage-dependent and voltage-independent channel gating.     

Social Network Analysis Predicts Health Behaviours and Self-Reported Health in African Villages

The provision of healthcare in rural African communities is a highly complex and largely unsolved problem. Two main difficulties are the identification of individuals that are most likely affected by disease and the prediction of responses to health interventions. Social networks have been shown to capture health outcomes in a variety of contexts. Yet, it is an open question as to what extent social network analysis can identify and distinguish among households that are most likely to report poor health and those most likely to respond to positive behavioural influences. We use data from seven highly remote, post-conflict villages in Liberia and compare two prominent network measures: in-degree and betweenness. We define in-degree as the frequency in which members from one household are named by another household as a friends. Betweenness is defined as the proportion of shortest friendship paths between any two households in a network that traverses a particular household. We find that in-degree explains the number of ill family members, whereas betweenness explains engagement in preventative health. In-degree and betweenness independently explained self-reported health and behaviour, respectively. Further, we find that betweenness predicts susceptibility to, instead of influence over, good health behaviours. The results suggest that targeting households based on network measures rather than health status may be effective for promoting the uptake of health interventions in rural poor villages.     

Genome-Wide Comparison of Cowpox Viruses Reveals a New Clade Related to Variola Virus

Zoonotic infections caused by several orthopoxviruses (OPV) like monkeypox virus or vaccinia virus have a significant impact on human health. In Europe, the number of diagnosed infections with cowpox viruses (CPXV) is increasing in animals as well as in humans. CPXV used to be enzootic in cattle; however, such infections were not being diagnosed over the last decades. Instead, individual cases of cowpox are being found in cats or exotic zoo animals that transmit the infection to humans. Both animals and humans reveal local exanthema on arms and legs or on the face. Although cowpox is generally regarded as a self-limiting disease, immunosuppressed patients can develop a lethal systemic disease resembling smallpox. To date, only limited information on the complex and, compared to other OPV, sparsely conserved CPXV genomes is available. Since CPXV displays the widest host range of all OPV known, it seems important to comprehend the genetic repertoire of CPXV which in turn may help elucidate specific mechanisms of CPXV pathogenesis and origin. Therefore, 22 genomes of independent CPXV strains from clinical cases, involving ten humans, four rats, two cats, two jaguarundis, one beaver, one elephant, one marah and one mongoose, were sequenced by using massive parallel pyrosequencing. The extensive phylogenetic analysis showed that the CPXV strains sequenced clearly cluster into several distinct clades, some of which are closely related to Vaccinia viruses while others represent different clades in a CPXV cluster. Particularly one CPXV clade is more closely related to Camelpox virus, Taterapox virus and Variola virus than to any other known OPV. These results support and extend recent data from other groups who postulate that CPXV does not form a monophyletic clade and should be divided into multiple lineages.     

Synthesis and derivatization of halflanthanidocene aryl(alk)oxide complexes

The reaction of halflanthanidocene aryloxides Cp^R′Ln(OAr^tBu,R)₂ (Ln = Y, La, Lu; Cp^R′ = C₅Me₅, C₄Me₄H; R = H, Me) and halflanthanidocene alkoxides [(C₅Me₅)Ln(OCH₂CMe₃)₂]₂ (Ln = Y, Lu) with trimethylaluminum (TMA) was investigated. Monomeric Cp^R′Ln(OAr^tBu,R)₂, derived from the ortho-tBu-substituted OC₆H₂tBu₂-2,6-R-4 (R = H, Me) ligands, form mono(tetramethylaluminate) complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) for the smaller lanthanide metal centers yttrium and lutetium. Such an [aryloxide] → [aluminate] ligand exchange was not observed at the larger lanthanum metal center. The mobility of the tetramethylaluminate ligands of complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) (Ln = Y, Lu) was examined by variable-temperature (VT) ¹H NMR spectroscopy, revealing two signals for bridging and terminal methyl groups at lower temperatures. The treatment of complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) with donor solvent d₈-THF gave Cp^R′Ln(OAr^tBu,R)(Me)(d₈-THF)₂ (Ln = Y, Lu) with terminal methyl groups, according to a donor-induced aluminate cleavage reaction. Dimeric [(C₅Me₅)Ln(OCH₂CMe₃)₂]₂ (Ln = Y, Lu) was synthesized from (C₅Me₅)Ln(NiPr₂)₂(THF) and reacted with two equivalents of TMA per Ln center to yield monomeric bis(TMA) adduct complexes (C₅Me₅)Ln(OCH₂CMe₃)₂(AlMe₃)₂(Ln = Y, Lu). VT NMR spectroscopic studies confirmed a high mobility of the Ln(μ-OCH₂CMe₃)(μ-Me)AlMe₂ moieties at an ambient temperature. Both bis(TMA) adduct complexes were characterized by X-ray structure analysis.