A Stochastic Model for Microtubule Motors Describes the In Vivo Cytoplasmic Transport of Human Adenovirus

Cytoplasmic transport of organelles, nucleic acids and proteins on microtubules is usually bidirectional with dynein and kinesin motors mediating the delivery of cargoes in the cytoplasm. Here we combine live cell microscopy, single virus tracking and trajectory segmentation to systematically identify the parameters of a stochastic computational model of cargo transport by molecular motors on microtubules. The model parameters are identified using an evolutionary optimization algorithm to minimize the Kullback-Leibler divergence between the in silico and the in vivo run length and velocity distributions of the viruses on microtubules. The present stochastic model suggests that bidirectional transport of human adenoviruses can be explained without explicit motor coordination. The model enables the prediction of the number of motors active on the viral cargo during microtubule-dependent motions as well as the number of motor binding sites, with the protein hexon as the binding site for the motors

Effects of N-Glycosylation Site Removal in Archaellins on the Assembly and Function of Archaella in Methanococcus maripaludis

In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation

Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly

Оптимальний підбір пластифікатора для асфальтобетонного покриття

Purpose. To investigate the optimal selection of plasticizer for asphalt concrete coatings.  The fragility of asphalt concrete, which occurs as a result of aging, exposure to sunlight, temperature variations, does not always allow it to be reused without plasticizers, which can affect the intensity of intermolecular interaction and, accordingly, the properties of asphalt concrete, create a system of elasticity.  The option of plasticizers in the course of laboratory studies was carried out in accordance with the natural qualities of bitumen and plasticizers. Methods. Comparison of the brittleness and strength dependences on the deformation rate. Results. The dependence of the temperature of brittleness and strength on the deformation rate is established. In the course of experiments, on a millimeter paper, a load diagram was written − a deflection using an inductive sensor connected to the test drive CD-10 through an amplifier, which allows to increase the actual deflection by 1000 times. Scientific novelty. The study of asphalt concrete for bending over a wide range of temperatures and deformation rates allowed to determine the temperature of brittleness of asphalt concrete after thermal aging. Practical relevance. Thus, the proposed method for reducing the monolithicity of asphalt concrete in the coating by introducing the optimal amount of plasticizer (lubricant). The dependence of the temperature of brittleness of  stressed asphalt concrete on the rate of deformation in the range of 15…544 mm/min is established. At temperatures from +50 to −20 °C it is possible to calculate their Tbr at any rate of bending according to the data obtained experimentally at two values of the bending rate. The difference in the value of Tbr of individual asphalt concrete in the specified range of bending rates reaches 30 °C.Постановка проблемы.Необходимо исследовать оптимальный подбор пластификатора для асфальтобетонного покрытия.  Хрупкость асфальтобетона, которая возникает в результате старения, воздействия солнечных лучей, перепада температур, невсегда позволяет его использовать повторно без пластификаторов, которые могут влиять на интенсивность межмолекулярного взаимодействия и, соответственно, на свойства асфальтобетона, делают систему эластичной.  Выбор пластификаторов в процессе лабораторных исследований проводился в зависимости от природных качеств битумов и пластификаторов. Методика. Сравнение зависимостей температур хрупкости и прочности от скорости деформирования. Результаты. Установлена зависимость температуры хрупкости и прочности от скорости деформирования. В процессе экспериментов записывали на миллиметровой бумаге диаграмму нагрузок ‑ прогиб с помощью индуктивного датчика, подключенного к испытательной машине СД-10 через усилитель, который позволяет увеличивать фактический прогиб в 1 000 раз. Научная новизна. Исследование асфальтобетона на изгиб в широком интервале температур и скорости деформирования позволило определить температуру хрупкости асфальтобетона после термостарения. Практическая значимость. Предложен способ восстановления монолитности асфальтобетона в покрытии путем введения оптимального количества пластификатора (смазки). Установленная зависимость температуры хрупкости напряженного асфальтобетона от скорости деформации в интервале 15…544 мм/мин.  при температуре от +50 до −20 0С позволяет рассчитать их Ткр при любой скорости сгибания по данным, полученным экспериментально при двух значениях скорости сгибания. Разница в величине Ткр отдельных асфальтобетонов в указанном интервале скоростей сгибания достигает 30 0С.Мета. Дослідити оптимальний підбір пластифікатора для асфальтобетонного покриття. Крихкість асфальтобетону, яка виникає в результаті старіння, дії сонячних променів, перепаду температур не завжди дозволяє його використовувати повторно без пластифікаторів, які можуть впливати на інтенсивність міжмолекулярної взаємодії і, відповідно на властивості асфальтобетону, створюють систему еластичною. Вибір пластифікаторів в процесі лабораторних досліджень проводився в залежності від природніх якостей бітумів і пластифікаторів. Методика. Порівняння залежностей температур крихкості і міцності від швидкості деформування. Результати. Встановлено залежність температури крихкості і міцності від швидкості деформування. В процессі експериментів записували на міліметровому папері діаграму навантажень – прогин за допомогою індуктивного датчика, підключеного до випробувальної машини ЦД-10 через посилювач, який дозволяє збільшувати фактичний прогин в 1 000 разів. Наукова новизна. Дослідження асфальтобетону на вигин в широкому інтервалі температур и швидкості деформування дозволили визначити температуру крихкості асфальтобетону після термостаріння. Практична значимість. Таким чином, запропонований спосіб відновлення монолітності асфальтобетону в покритті шляхом введення оптимальної кількості пластифікатора (мастила). Встановлена залежність температури крихкості напруженого асфальтобетону від швидкості деформації в інтервалі 15…544 мм/хв. за температурою від +50 до –20 0С дозволяє розрахувати їх Ткр при будь-якій швидкості згинання за даними, отриманими експериментально при двох значеннях швидкості згинання. Різниця у величині Ткр  окремих асфальтобетонів у вказаному інтервалі швидкостей згинання досягає 30 0С

Complementation of an <i>aglB</i> Mutant of <i>Methanococcus maripaludis</i> with Heterologous Oligosaccharyltransferases

<div><p>The oligosaccharyltransferase is the signature enzyme for <i>N</i>-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in <i>Methanococcus maripaludis</i> deleted for <i>aglB</i>, using archaellin FlaB2 as the reporter protein since all archaellins in <i>Mc</i>. <i>maripaludis</i> are modified at multiple sites by an <i>N</i>-linked tetrasaccharide and this modification is required for archaellation. In the <i>Mc</i>. <i>maripaludis</i> Δ<i>aglB</i> strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from <i>Methanococcus voltae</i> and <i>Methanothermococcus thermolithotrophicus</i> functionally replaced the oligosaccharyltransferase activity missing in the <i>Mc</i>. <i>maripaludis</i> Δ<i>aglB</i> strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from <i>Mc</i>. <i>voltae</i> has a relaxed specificity for the linking sugar of the transferred glycan since while the <i>N</i>-linked glycan present in <i>Mc</i>. <i>voltae</i> is similar to that of <i>Mc</i>. <i>maripaludis</i>, the <i>Mc</i>. <i>voltae</i> glycan uses <i>N</i>-acetylglucosamine as the linking sugar. In <i>Mc</i>. <i>maripaludis</i> that role is held by <i>N</i>-acetylgalactosamine. This study also identifies <i>aglB</i> from <i>Mtc</i>. <i>thermolithotrophicus</i> for the first time by its activity. Attempts to use AglB from <i>Methanocaldococcus jannaschii</i>, <i>Haloferax volcanii</i> or <i>Sulfolobus acidocaldarius</i> to functionally replace the oligosaccharyltransferase activity missing in the <i>Mc</i>. <i>maripaludis</i> Δ<i>aglB</i> strain were unsuccessful.</p></div

Primers used in this study.

<p>Primers used in this study.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Comparison of <i>Mc</i>. <i>maripaludis</i> AglB to other AglBs used in this study (pairwise EMBOSS Needle).

<p>Comparison of <i>Mc</i>. <i>maripaludis</i> AglB to other AglBs used in this study (pairwise EMBOSS Needle).</p

Western blot analysis of FlaB2 in an <i>Mc</i>. <i>maripaludis</i> Δ<i>aglB</i>-<i>14-9</i> mutant complemented <i>in trans</i> with homologous <i>aglB</i>.

<p>The Δ<i>aglB-14-9</i> mutant was complemented with a plasmid borne version of <i>Mc</i>. <i>maripaludis aglB</i> under the control of the <i>nif</i> promoter. The complemented Δ<i>aglB-14-9</i> mutant was grown in nitrogen-free medium supplemented with alanine or NH₄Cl which results in transcription from the <i>nif</i> promoter being on (alanine) or off (NH₄Cl). Lane 1 is Mm900 (WT), lane 2 is Δ<i>aglB-14-9</i> mutant, lane 3 is Δ<i>aglB-14-9</i> mutant complemented cells grown in nitrogen-free medium supplemented with alanine, lane 4 is Δ<i>aglB-14-9</i> mutant complemented cells grown in nitrogen-free medium supplemented with NH₄Cl.</p