8 research outputs found
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High-efficacy subcellular micropatterning of proteins using fibrinogen anchors.
Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology
Coarse-grained simulations of actomyosin rings point to a nodeless model involving both unipolar and bipolar myosins
Cytokinesis in many eukaryotic cells is orchestrated by a contractile actomyosin ring. While many of the proteins involved are known, the mechanism of constriction remains unclear. Informed by the existing literature and new three-dimensional (3D) molecular details from electron cryotomography, here we develop 3D coarse-grained models of actin filaments, unipolar and bipolar myosins, actin cross-linkers, and membranes and simulate their interactions. Assuming that local force on the membrane results in inward growth of the cell wall, we explored a matrix of possible actomyosin configurations and found that node-based architectures like those presently described for ring assembly result in membrane puckers not seen in electron microscope images of real cells. Instead, the model that best matches data from fluorescence microscopy, electron cryotomography, and biochemical experiments is one in which actin filaments transmit force to the membrane through evenly distributed, membrane-attached, unipolar myosins, with bipolar myosins in the ring driving contraction. While at this point this model is only favored (not proven), the work highlights the power of coarse-grained biophysical simulations to compare complex mechanistic hypotheses
Structure of the fission yeast actomyosin ring during constriction
Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of âŒ150 nm in diameter that âsaddlesâ the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane
Structure of the fission yeast actomyosin ring during constriction
Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of âŒ150 nm in diameter that âsaddlesâ the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane
GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements
Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5â2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility
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In vitro reconstitution of Central Spindle Motility
Precise localisation of intracellular organelles impacts a myriad of cellular processes including cell motility, polarisation and mitosis. Positioning is actively controlled by organelle specific attachment, often involving dedicated motor proteins, to cytoskeletal network. While much is known about the biophysics of motors on isolated tracks, how the complex cytoskeleton topologies found in cells, such as antiparallel microtubule (MT) overlaps, can influence the steady state distribution of cargoes is not understood molecularly. For example, in dividing sensory organ precursor cells, the antiparallel MT overlap of the central spindle controls the asymmetric segregation of signalling endosomes containing fate determinants in only one daughter cell. A modest asymmetry in the density of microtubules within the antiparallel MT overlap of central spindle, with more MTs on the anterior side of the spindle, generates one order of magnitude higher (than the microtubule asymmetry) bias distribution of endosomes. How such moderate asymmetry in the cytoskeleton is translated by motor proteins to produce strong non-linear effects and how different endosomes respond to this asymmetry is unknown.
In this work, I have aimed to address both these questions by using a combination of in vivo imaging and in vitro reconstitution using purified proteins and micropatterning.
To address how different endosomes segregate on an asymmetric microtubule track I utilised knock-in Rab-GFP lines and internalised labelled anti-delta antibodies. Remarkably, a class of late endosomes containing anti-delta antibodies indeed partitioned symmetrically during mitosis even on an asymmetric central spindle. This suggested that endosome partitioning is dictated by both the microtubule track and the endosome specific motor proteins in vivo.
To gain insight into how the motor content affects endosome behaviour, particularly on defined microtubule network, I attempted to reconstitute the process in vitro. The central spindle, an interdigitated antiparallel microtubule structure, is formed by balanced activity between both motor and non-motor microtubule associating proteins (MAPS). To control the geometry of such antiparallel microtubule tracks I utilised an improved micropatterning technology and purified central spindle proteins. This combined approach successfully led to formation of asymmetric antiparallel overlaps akin to the central spindle for the first time
Predictability of temporal variation in climate and the evolution of seasonal polyphenism in tropical butterfly communities
Phenotypic plasticity in heterogeneous environments can provide tight environment-phenotype matching. However, the prerequisite is a reliable environmental cue(s) that enables organisms to use current environmental information to induce the development of a phenotype with high fitness in a forthcoming environment. Here, we quantify predictability in the timing of precipitation and temperature change to examine how this is associated with seasonal polyphenism in tropical Mycalesina butterflies. Seasonal precipitation in the tropics typically results in distinct selective environments, the wet and dry seasons, and changes in temperature can be a major environmental cue. We sampled communities of Mycalesina butterflies from two seasonal locations and one aseasonal location. Quantifying environmental predictability using wavelet analysis and Colwell's indices confirmed a strong periodicity of precipitation over a 12-month period at both seasonal locations compared to the aseasonal one. However, temperature seasonality and periodicity differed between the two seasonal locations. We further show that: (a) most females from both seasonal locations synchronize their reproduction with the seasons by breeding in the wet season but arresting reproduction in the dry season. In contrast, all species breed throughout the year in the aseasonal location and (b) species from the seasonal locations, but not those from the aseasonal location, exhibited polyphenism in wing pattern traits (eyespot size). We conclude that seasonal precipitation and its predictability are primary factors shaping the evolution of polyphenism in Mycalesina butterflies, and populations or species secondarily evolve local adaptations for cue use that depend on the local variation in the environment.Peer reviewe