Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

Twenty years of traffic: A 2020 vision of cellular electron microscopy

The last 20 years have seen incredible advances in electron microscopy (EM). Cryoelectron microscopy can now resolve protein structures to a previously unimaginable resolution but the advances in cellular EM are just as significant. I will take this opportunity to briefly summarize some of the new developments in cellular EM.     

Dosimetric effect of intrafraction motion and different localization strategies in prostate SBRT

The aim of this study was to evaluate the dosimetric effect of continuous motion monitoring based localization (Calypso, Varian Medical Systems), gating and intrafraction motion correction in prostate SBRT. Delivered doses were modelled by reconstructing motion inclusive dose distributions for different localization strategies. Actually delivered dose (strategy A) utilized initial Calypso localization, CBCT and additional pre-treatment motion correction by kV-imaging and Calypso, and gating during the irradiation. The effect of gating was investigated by simulating non-gated treatments (strategy B). Additionally, non-gated and single image-guided (CBCT) localization was simulated (strategy C). A total of 308 fractions from 22 patients were reconstructed. The dosimetric effect was evaluated by comparing motion inclusive target and risk organ dose-volume parameters to planned values. Motion induced dose deficits were seen mainly in PTV and CTV to PTV margin regions, whereas CTV dose deficits were small in all strategies: mean ± SD difference in CTVD99% was –0.3 ± 0.4%, −0.4 ± 0.6% and –0.7 ± 1.2% in strategies A, B and C, respectively. Largest dose deficits were seen in individual fractions for strategy C (maximum dose reductions were −29.0% and –7.1% for PTVD95% and CTVD99%, respectively). The benefit of gating was minor, if additional motion correction was applied immediately prior to irradiation. Continuous motion monitoring based localization and motion correction ensured the target coverage and minimized the OAR exposure for every fraction and is recommended to use in prostate SBRT. The study is part of clinical trial NCT02319239.     

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cβ/G protein pathway

Some proteins can serve multiple functions depending on different cellular conditions. An example of a bifunctional protein is inositide‐specific mammalian phospholipase Cβ (PLCβ). PLCβ is activated by G proteins in response to hormones and neurotransmitters to increase intracellular calcium. Recently, alternate cellular function(s) of PLCβ have become uncovered. However, the conditions that allow these different functions to be operative are unclear. Like many mammalian proteins, PLCβ has a conserved catalytic core along with several regulatory domains. These domains modulate the intensity and duration of calcium signals in response to external sensory information, and allow this enzyme to inhibit protein translation in a noncatalytic manner. In this review, we first describe PLCβ's cellular functions and regulation of the switching between these functions, and then discuss the thermodynamic considerations that offer insight into how cells manage multiple and competitive associations allowing them to rapidly shift between functional states.     

植物原生质体在分子细胞生物学研究中的应用

植物原生质体是去除了细胞壁的裸露细胞,其具有细胞全能性,现广泛应用于植物分子细胞生物学的研究中,可以大大缩减实验周期,并有助于得到体内实验的实时检测数据。该文除了介绍植物原生质体的提取和纯化方法外,还对国内外利用各种植物的原生质体进行细胞瞬时转化、亚细胞定位、细胞融合和大分子复合物相互作用等试验进行了总结和讨论。植物原生质体还可用于基因表达模式的实时检测,并作为生物反应器的受体细胞进行代谢物的体外生产。此外,还对当前该技术所面临的瓶颈进行了分析,为植物原生质体在分子细胞生物学领域的应用提供帮助,为技术的优化和推广提供参考。     

小麦TaCO9基因的克隆及功能分析

为了明确TaCO9-1A基因在小麦生长发育中的具体调控机理,该研究以同源克隆的方法成功获得了大麦(Hordeum vulgare)光周期基因HvCO9在小麦(Triticum aestivum)中的直系同源基因TaCO9,并对其进行生物信息学分析、亚细胞定位、转录激活以及表达模式分析;利用农杆菌侵染法转化拟南芥,并对过表达株系进行表型分析。结果表明:(1)TaCO9包含2个外显子和1个内含子,CDS区全长876 bp,编码291个氨基酸,蛋白序列含有特有的CCT结构域,且在不同物种间高度保守。(2)生物信息学分析表明,TaCO9基因编码的蛋白质分子量约为30.7 kD,等电点为6.24;TaCO9的启动子区含有光响应、激素应答和胁迫应答等多种顺式作用元件。(3)亚细胞定位和转录活性分析表明,TaCO9主要定位于细胞核中,且具有转录激活活性。(4)qRT-PCR结果表明,TaCO9基因在各个组织中均有表达,叶片中的表达量最高;在光照14 h条件下TaCO9的表达量显著高于光照10 h和12 h条件下的表达量,且TaCO9在大粒小麦‘西农817’子房与籽粒中的表达量显著高于‘中国春’。(5)经潮霉素筛选成功获得3个转基因拟南芥株系;进一步功能鉴定结果表明,过表达转TaCO9基因拟南芥植株的开花期迟于野生型(对照)3~4 d,但角果和籽粒较野生型大。     

Acoustic localization of terrestrial wildlife: Current practices and future opportunities

Autonomous acoustic recorders are an increasingly popular method for low‐disturbance, large‐scale monitoring of sound‐producing animals, such as birds, anurans, bats, and other mammals. A specialized use of autonomous recording units (ARUs) is acoustic localization, in which a vocalizing animal is located spatially, usually by quantifying the time delay of arrival of its sound at an array of time‐synchronized microphones. To describe trends in the literature, identify considerations for field biologists who wish to use these systems, and suggest advancements that will improve the field of acoustic localization, we comprehensively review published applications of wildlife localization in terrestrial environments. We describe the wide variety of methods used to complete the five steps of acoustic localization: (1) define the research question, (2) obtain or build a time‐synchronizing microphone array, (3) deploy the array to record sounds in the field, (4) process recordings captured in the field, and (5) determine animal location using position estimation algorithms. We find eight general purposes in ecology and animal behavior for localization systems: assessing individual animals' positions or movements, localizing multiple individuals simultaneously to study their interactions, determining animals' individual identities, quantifying sound amplitude or directionality, selecting subsets of sounds for further acoustic analysis, calculating species abundance, inferring territory boundaries or habitat use, and separating animal sounds from background noise to improve species classification. We find that the labor‐intensive steps of processing recordings and estimating animal positions have not yet been automated. In the near future, we expect that increased availability of recording hardware, development of automated and open‐source localization software, and improvement of automated sound classification algorithms will broaden the use of acoustic localization. With these three advances, ecologists will be better able to embrace acoustic localization, enabling low‐disturbance, large‐scale collection of animal position data.     

核定位信号及其在病毒感染机制中的研究进展

核定位信号介导的蛋白入核是细胞内信号传递网络中核内外物质信息交流的重要一环,绝大多数病毒蛋白进入细胞核均需要核质转运受体识别和结合入核蛋白携带的核定位信号序列。病毒蛋白的入核转运机制在病毒感染过程中起着至关重要的作用,对于病毒的复制、毒力具有重要意义,针对该机制的研究有利于新的抗病毒靶点的发现。本文对核定位信号的分类信息进行了总结,并对不同的核质转运受体及其介导的入核机制进行了比较分析,概述了病毒入核蛋白及其核定位信号在病毒感染机制中的研究发现。     

荻草谷网蚜唾液蛋白基因Sm13498的克隆及功能分析

【目的】荻草谷网蚜Sitobion miscanthi为我国小麦Triticum aestivum主产区麦蚜优势种;Sm13498蛋白是在荻草谷网蚜唾液腺中特异表达的唾液蛋白。本研究旨在探析荻草谷网蚜功能未知的Sm13498在调节植物防御反应中的潜在作用。【方法】基于荻草谷网蚜唾液腺转录组测序数据,PCR克隆Sm13498的cDNA全长序列,并进行生物信息学分析;采用RT-qPCR测定Sm13498在取食小麦叶片不同时间的荻草谷网蚜无翅成蚜中的表达动态;通过酵母分泌系统验证Sm13498蛋白信号肽的分泌功能;利用根癌农杆菌Agrobacterium tumefaciens介导在本氏烟Nicotiana benthamiana中瞬时表达技术鉴定Sm13498蛋白功能及亚细胞定位。【结果】克隆获得了荻草谷网蚜Sm13498 cDNA全长序列(GenBank登录号：MW346655),开放阅读框(ORF)全长783 bp,编码260个氨基酸,预测蛋白分子量28.01 kD,第1-22位氨基酸为N端信号肽。系统进化树显示,Sm13498与豌豆蚜Acyrthosiphon pisum的功能未知蛋白LOC100159087precursor(GenBank登录号: NP_001313548.1)亲缘关系最近(氨基酸序列一致性为71.7%)。RT-qPCR结果表明,Sm13498在荻草谷网蚜无翅成蚜取食小麦叶片12 h时表达水平达到最高。含有Sm13498信号肽片段的酿酒酵母Saccharomyces cerevisiae YTK12可在YPRAA培养基正常生长,并可将无色2,3,5-氯化三苯基四氮唑(TTC)还原为不可溶的暗红色的氯化三苯基四氮唑(TTF),证实其信号肽具有分泌活性。经根癌农杆菌介导在本氏烟瞬时表达Sm13498蛋白可抑制Bcl-2相关X蛋白(Bcl-2-associated X protein, BAX)及病原菌激发子INF1诱导的程序性细胞死亡。亚细胞定位结果表明,Sm13498-GFP融合蛋白定位于本氏烟叶片细胞膜。【结论】结果说明荻草谷网蚜唾液蛋白Sm13498可抑制植物防御反应。本研究为发掘荻草谷网蚜唾液中效应子, 深入解析麦蚜对小麦品种强适应性奠定了基础。