The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

The vertex specific proteins pUL17 and pUL25 mechanically reinforce Herpes Simplex Virus capsids

Using atomic force microscopy imaging and nanoindentation measurements, we investigated the effect of the minor capsid proteins pUL17 and pUL25 on the structural stability of the icosahedral Herpes Simplex Virus capsids. pUL17 and pUL25 that form the capsid vertex-specific component (CVSC) particularly contributed to the capsid resilience along the 5-fold and 2-fold, but not along the 3-fold icosahedral axes. Our detailed analyses, including quantitative mass spectrometry on the protein composition of the capsids, revealed that pUL17 and pUL25 are both required to stabilize the capsid shells at the vertices. This indicates that herpesviruses withstand the internal pressure that is generated during DNA genome packaging by locally reinforcing the mechanical sturdiness of the vertices, the most stressed part of the capsids.IMPORTANCE In this study the structural, material properties of Herpes Simplex Virus type 1 were investigated. The capsid of Herpes Simplex Virus is built up of a variety of proteins and we scrutinized the influence of two of these proteins on the stability of the capsid. For this we used a scanning force microscope that makes detailed, topographic images of the particles and that is able to perform mechanical deformation measurements. Using this approach we revealed that both studied proteins play an essential role in viral stability. These new insights support us to form a complete view on viral structure and could furthermore possibly not only help to develop specific anti-virals, but also to build protein shells with improved stability for drug delivery purposes

The chaperone dynein LL1 mediates cytoplasmic transport of empty and mature hepatitis B virus capsids

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Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

<div><p>Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.</p></div

Quantification of fluorescence intensity of the markers microinjected with or without H1 into U2OS cells.

<p>Quantifications of the fluorescences presented as videos in the supporting information. The microinjected markers are indicated on top of each panel (D10-Tx: Dextran 10-Texas Red labelled, D40-FITC: Dextran 40, FITC-labelled, ab-A647: unrelated IgG, Alexa.647-labelled). The grey lines show the cytoplasmic, the red line the nuclear fluorescence. The y-axis depicts the intensity given in arbitrary units, the x-axis the time after microinjection in seconds. <b>A.</b> Microinjection of buffer with marker proteins. D10-Tx equilibrated between cytoplasm and nucleus directly after microinjection while D40-FITC and ab-A647 stayed excluded from nucleus. <b>B.</b> Microinjection of H1 with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 240 seconds after microinjection and reached the equilibrium. <b>C.</b> Microinjection of Ca⁺⁺ with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 200 seconds after microinjection also reaching the equilibrium. In summery the panels show that both H1 and Ca⁺⁺ triggered sudden NEBD approx. 2–3 min after microinjection. Dextran 40 and the antibodies entered the nucleus at the same time indicating a catastrophic-like destruction of the barrier as it was also seen for nuclear escape of 100 kDa cargos and chromatin in permeabilized cells.</p

NEBD capacity of different PV.

<p>Per permeabilized HeLa cell 300 of each PV were added. The figure shows the quantification of the PI stain as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003671#ppat-1003671-g002" target="_blank">figure 2B</a>, and the 95% CIs at each time point. Red dotted line: buffer only (n = 14). Blue line: AAV2 (n = 13), orange line: canine parvovirus (n = 35), red line: H1 (n = 23), black line: AAV2 after acidification to pH 5.2 and subsequent neutralization (n = 10). Acidification to pH 5.2 of H1 did not change the NEBD activity (not shown). The figure shows that NEBD is independent upon the type of PV with a similar kinetic but to a different extent. Acidification was needed for AAV but not for H1 and the canine parvovirus.</p

Intra and inter assay variability of chromatin loss in permeabilized HeLa cells incubated with or without H1.

<p>Merge of six experiments. The chromatin by PI was quantified in 42 nuclei exposed to H1 and in 67 nuclei exposed to buffer only. <b>A.</b> Each circle represents the relative PI fluorescence at the indicated time. The fluorescence at t = 0 of each cell was set as 100%. The lines connect the fluorescence of one nucleus at different times. Each colour represents one experiment. Y-axis: relative fluorescence in %, x-axis: time in min. <b>B.</b> Same data as in A. but showing the mean fluorescence and the 95% confidence intervals (CI) (bars). The horizontal bar shows the 95% CI for the 50% loss of PI fluorescence (4.6 to 5.4 min). The data show that parvovirus-mediated NEBD exhibits small variations between different assays and between individual nuclei.</p

Cellular PKC and cdk-2 become activated by H1 and PKCα but not PKCβ is required for NEBD.

<p><b>A, B, C:</b> Activation of PKC, cdk-2 and caspase-3 in permeabilized HeLa cells by H1. Y-axis: activity in arbitrary units. The tested activity is indicated on top of each panel. The columns show the mean values of three independent experiments. The variation bars show the range between the highest and the lowest value. 1. permeabilized cells. 2. permeabilized cells+H1. 3. permeabilized cells+H1+H89. 4. permeabilized cells+H1+Roscovitine. 5. permeabilized cells+H1+zVAD-fmk. 6. H1 without cells. 7. permeabilized cells+H1+thapsigargin. 8. non-permeabilized cells. n.d. not determined. The panels show that permeabilization decrease the activity of all three enzymes and that H1 activates the activities of PKC and cdk-2, while an effect on caspase-3 is doubtful. Inhibition of PKC also reduced activity of cdk2 but not of caspase_3 despite of its inhibitor specificity shown in the supporting information. Thapsigargin pre-treatment, leading to Ca⁺⁺ depletion inhibits PKC and cdk2 implying that a Ca⁺⁺-dependent PKC is involved. <b>D.</b> PV H1-mediated NEBD is inhibited by PKCα but not PKCβ. Quantification of PI-stained chromatin of permeabilized HeLa cells to which 300 H1 per permeabilized cell were added. The bars depict 95% CI. Red, dotted line: buffer (n = 28); red line: H1 (n = 14); green dashed line: H1 using PKCα-inhibited cells (n = 19); black dashed line: H1 using PKCβ-inhibited cells. Collectively, the data show that Ca⁺⁺-dependent PKCα is required for NEBD, which is consistent with the PV-mediated activation of a Ca⁺⁺-dependent PKC. PKCα subsequently activated cdk-2, which was also shown essential for NEBD. Caspase-3 was not significantly activated by PV but its activity was however essential for NEBD.</p

PV interact directly with nucleoporins required for NEBD.

<p><b>A.</b> WGA does not inhibit NEBD upon addition of 300 H1 per permeabilized cell. The figure shows the quantification of the PI stain as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003671#ppat-1003671-g002" target="_blank">figure 2B</a>, and the 95% CIs at each time point. Red dotted line: buffer only (n = 8), red line: H1, green dashed line: H1 in the presence of 1 mg/ml WGA (n = 4). <b>B.</b> Parvoviruses bind directly to nucleoporins. Co-precipitated Nups were detected by Western blot using the mAb414, which interacts with different FG repeat-containing Nups including Nup358, 214,153 and 62. The MW is given on the left, the Nups are indicated on the right. 1: AAV2 (acidified and neutralized) without Nups, 2: same AAV2+Nups, 3: H1, 4: H1+Nups, 5: beads+Nups, 6: 12 µg, 7: 36 µg Nups directly loaded on the gel. Nup153 migrates at ∼170 kDa, as it was described elsewhere <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003671#ppat.1003671-Sukegawa1" target="_blank">[70]</a>. <b>C.</b> Blocking the NPCs by hepatitis B virus capsids inhibits H1-mediated NEBD. Conditions and read-out as in A. Red dotted line: buffer only (n = 14), red dashed line: H1 after pre-incubation of the NPCs with an excess (1200 ng) of <i>in vitro</i> phosphorylated capsids of the hepatitis B virus in the presence of transport receptors (n = 18), green dashed line: same treatment with a mutant of the hepatitis B virus capsid, which lacks the C terminus that is need for NPC interaction (n = 9), red line: H1 (n = 7).</p

H1-mediated NEBD leads to simultaneous escape of a 100 kDa cargo and chromatin, which depends upon enzymes needed for mitosis.

<p>Nuclei of permeabilized cells preloaded with M9-Alexa647-BSA (M9) prior to addition of 300 H1 per permeabilized HeLa cell together with inhibitors. The graphs show quantifications of DAPI and Alexa647 fluorescences with the mean values and CI 95% (bars). Blue dotted line: DAPI with buffer only, no inhibitor (n = 39), pink dotted line: M9, buffer, no inhibitor (n = 39), orange line: DAPI, H1, no inhibitor (n = 21), red line: M9, H1, no inhibitor (n = 21), black line: DAPI, H1, H89 (n = 33), brown blue dashed line: M9, H1, H89 (n = 33), blue dashed line: DAPI, H1, roscovitine (n = 19), grey dashed line: M9, H1, roscovitine (n = 19), cyan dashed line: DAPI, H1, zVAD-fmk (n = 23), grey: M9, H1, zVAD-fmk (n = 23). Please note that some lines overlap. The figure shows that the escape of chromatin and the 100 kDa cargo cannot be separated despite of their different MW, indicating a catastrophic-like event during which the entire NE disintegrates. As all inhibitors entirely inhibited the loss of both cargos the results further indicate that PKC, cdks and caspase-3 are essential for NEBD.</p