Phase transition of spindle-associated protein regulate spindle apparatus assembly.

Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks.

Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitate vertebrate blastula development.

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m6 A, but repressed by m5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.

Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling.

Cilia of higher animals sense various environmental stimuli. Proper ciliary signaling requires appropriate extent of BBSome-mediated export of membrane receptors across ciliary barrier transition zone (TZ) through retrograde intraflagellar transport (IFT) machinery. How the barrier passage is controlled, however, remains unknown. Here, we show that small GTPase Rabl2 functions as a molecular switch for the outward TZ passage. Rabl2-GTP enters cilia by binding to IFT-B complex. Its GTP hydrolysis enables the outward TZ passage of the BBSome and its cargos with retrograde IFT machinery, whereas its persistent association leads to their shedding from IFT-B during the passing process and consequently ciliary retention. Rabl2 deficiency or expression of a GTP-locked mutant impairs the ciliary hedgehog signaling without interfering with ciliation and respectively results in different spectrums of mouse developmental disorders. We propose that the switch role of Rabl2 ensures proper turnover of the BBSome and ciliary membrane receptors to fine-tune cilia-dependent signaling for normal embryonic development and organismic homeostasis.

Self-construction of actin networks through phase separation-induced abLIM1 condensates.

The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference results in sparse, poorly interconnected cortical actin networks and severe blebbing of migrating cells. Its isoforms, abLIM-L, abLIM-M, and abLIM-S, contain, respectively four, three, and no LIM domains, followed by a C terminus entirely homologous to erythroid cortex protein dematin. How abLIM1 functions, however, remains unclear. Here we show that abLIM1 is a liquid-liquid phase separation (LLPS)-dependent self-organizer of actin networks. Phase-separated condensates of abLIM-S-mimicking ΔLIM or the major isoform abLIM-M nucleated, flew along, and cross-linked together actin filaments (F-actin) to produce unique aster-like radial arrays and interconnected webs of F-actin bundles. Interestingly, ΔLIM condensates facilitated actin nucleation and network formation even in the absence of Mg2+. Our results suggest that abLIM1 functions as an LLPS-dependent actin nucleator and cross-linker and provide insights into how LLPS-induced condensates could self-construct intracellular architectures of high connectivity and plasticity.

Controlling Nucleation and Crystallization of CsPbI3 Perovskites for Efficient Inverted Solar Cells.

The unfavorable morphology and high crystallization temperature (Tc) of inorganic perovskites pose a significant challenge to their widespread application in photovoltaics. In this study, an effective approach is proposed to enhance the morphology of cesium lead triiodide (CsPbI3) while lowering its Tc. By introducing dimethylammonium acetate into the perovskite precursor solution, a rapid nucleation stage is facilitated, and significantly enhances the crystal growth of the intermediate phase at low annealing temperatures, followed by a slow crystal growth stage at higher annealing temperatures. This results in a uniform and dense morphology in CsPbI3 perovskite films with enhanced crystallinity, simultaneously reducing the Tc from 200 to 150 °C. Applying this approach in positive-intrinsic-negative (p-i-n) inverted cells yields a high power conversion efficiency of 19.23%. Importantly, these cells exhibit significantly enhanced stability, even under stress at 85 °C.

Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM.

The radial spoke (RS) heads of motile cilia and flagella contact projections of the central pair (CP) apparatus to coordinate motility, but the morphology is distinct for protozoa and metazoa. Here we show the murine RS head is compositionally distinct from that of Chlamydomonas Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a and Rsph10b, whose orthologs exist in the protozoan RS head. We resolve its cryo-electron microscopy (cryo-EM) structure at 3.2-Å resolution. Our atomic model further reveals a twofold symmetric brake pad-shaped structure, in which Rsph4a and Rsph9 form a compact body extended laterally with two long arms of twisted Rsph1 ß-sheets and potentially connected dorsally via Rsph3b to the RS stalk. Furthermore, our modeling suggests that the core complex contacts the periodic CP projections either rigidly through its tooth-shaped Rsph4a regions or elastically through both arms for optimized RS-CP interactions and mechanosignal transduction.

Peste des Petits Ruminants Virus Upregulates STING to Activate ATF6-Mediated Autophagy.

Peste des petits ruminants virus (PPRV) infection leads to autophagy, and the molecular mechanisms behind this phenomenon are unclear. Here, we demonstrate that PPRV infection results in morphological changes of the endoplasmic reticulum (ER) and activation of activating transcription factor 6 (ATF6) of the ER stress unfolded protein response (UPR). Knockdown of ATF6 blocked the autophagy process, suggesting ATF6 is necessary for PPRV-mediated autophagy induction. Further study showed that PPRV infection upregulates expression of the ER-anchored adaptor protein stimulator of interferon genes (STING), which is well-known for its pivotal roles in restricting DNA viruses. Knockdown of STING suppressed ATF6 activation and autophagy induction, implying that STING functions upstream of ATF6 to induce autophagy. Moreover, the STING-mediated autophagy response originated from the cellular pattern recognition receptor melanoma differentiation-associated gene 5 (MDA5). The absence of MDA5 abolished the upregulation of STING and the activation of autophagy. The deficiency of autophagy-related genes (ATG) repressed the autophagy process and PPRV replication, while it had no effect on MDA5 or STING expression. Overall, our work revealed that MDA5 works upstream of STING to activate ATF6 to induce autophagy. IMPORTANCEPPRV infection induces cellular autophagy; however, the intracellular responses and signaling mechanisms that occur upon PPRV infection are obscure, and whether innate immune responses are linked with autophagy to regulate viral replication is largely unknown. Here, we uncovered that the innate immune sensor MDA5 initiated the signaling cascade by upregulating STING, which is best known for its role in anti-DNA virus infection by inducing interferon expression. We first provide evidence that STING regulates PPRV replication by activating the ATF6 pathway of unfolded protein responses (UPRs) to induce autophagy. Our results revealed that in addition to mediating responses to foreign DNA, STING can cross talk with MDA5 to regulate the cellular stress response and autophagy induced by RNA viruses; thus, STING works as an adaptor protein for cellular stress responses and innate immune responses. Modulation of STING represents a promising approach to control both DNA and RNA viruses.

ECM1 controls T(H)2 cell egress from lymph nodes through re-expression of S1P(1).

Type 2 helper T cells (T(H)2) are critically involved in allergies and asthma. Here we demonstrate that extracellular matrix protein-1 (ECM1) is highly and selectively expressed in T(H)2 cells. ECM1 deficiency caused impaired T(H)2 responses and reduced allergic airway inflammation in vivo. Functional analysis demonstrated that although the T(H)2 polarization of ECM1-deficient cells was unimpaired, these cells had a defect in migration and were retained in peripheral lymphoid organs. This was associated with reduced expression of KLF2 and S1P(1). We also found that ECM1 could directly bind the interleukin-2 (IL-2) receptor to inhibit IL-2 signaling and activate S1P(1) expression. Our data identify a previously unknown function of ECM1 in regulating T(H)2 cell migration through control of KLF2 and S1P(1) expression.

Quantitative analysis of acetylation in peste des petits ruminants virus-infected Vero cells.

BACKGROUND: Peste des petits ruminants virus (PPRV) is a highly contagious pathogen that strongly influences the productivity of small ruminants worldwide. Acetylation is an important post-translational modification involved in regulation of multiple biological functions. However, the extent and function of acetylation in host cells during PPRV infection remains unknown. METHODS: Dimethylation-labeling-based quantitative proteomic analysis of the acetylome of PPRV-infected Vero cells was performed. RESULTS: In total, 1068 proteins with 2641 modification sites were detected in response to PPRV infection, of which 304 differentially acetylated proteins (DAcPs) with 410 acetylated sites were identified (fold change < 0.83 or > 1.2 and P < 0.05), including 109 up-regulated and 195 down-regulated proteins. Gene Ontology (GO) classification indicated that DAcPs were mostly located in the cytoplasm (43%) and participated in cellular and metabolic processes related to binding and catalytic activity. Functional enrichment indicated that the DAcPs were involved in the minichromosome maintenance complex, unfolded protein binding, helicase activity. Only protein processing in endoplasmic reticulum pathway was enriched. A protein-protein interaction (PPI) network of the identified proteins further indicated that a various chaperone and ribosome processes were modulated by acetylation. CONCLUSIONS: To the best of our knowledge, this is the first study on acetylome in PPRV-infected host cell. Our findings establish an important baseline for future study on the roles of acetylation in the host response to PPRV replication and provide novel insights for understanding the molecular pathological mechanism of PPRV infection.

Creation of poxvirus expressing foot-and-mouth and peste des petits ruminant disease virus proteins.

OBJECTIVE: Foot-and-mouth disease (FMD) and Peste des petits ruminant disease (PPR) are acute and severe infectious diseases of sheep and are listed as animal diseases for compulsory immunization. However, there is no dual vaccine to prevent these two diseases. The Modified Vaccinia virus Ankara strain (MVA) has been widely used in the construction of recombinant live vector vaccine because of its large capacity of foreign gene, wide host range, high safety, and immunogenicity. In this study, MVA-GFP recombinant virus skeleton was used to construct dual live vector vaccines against FMD and PPR. METHODS: The recombinant plasmid pUC57-FMDV P1-2A3CPPRV FH was synthesized and transfected into MVA-GFP infected CEF cells for homologous recombination. RESULTS: The results showed that a recombinant virus without fluorescent labeling was obtained after multiple rounds of plaque screening. The recombinant virus successfully expressed the target proteins, and the empty capsid of FMDV could be observed by transmission electron microscope (TME), and the expression levels of foreign proteins (VP1 and VP3) detected by ELISA were like those detected in FMDV-infected cells. This study laid the foundation for the successful construction of a live vector vaccine against FMD and PPR. KEY POINTS: • A recombinant MVA expressing FMDVP12A3C and PRRV HF proteins • Both the FMDV and PRRV proteins inserted into the virus were expressed • The proteins expressed by the recombinant poxvirus were assembled into VLPs.

Cep57 and Cep57l1 function redundantly to recruit the Cep63-Cep152 complex for centriole biogenesis.

The Cep63-Cep152 complex located at the mother centriole recruits Plk4 to initiate centriole biogenesis. How the complex is targeted to mother centrioles, however, is unclear. In this study, we show that Cep57 and its paralog, Cep57l1, colocalize with Cep63 and Cep152 at the proximal end of mother centrioles in both cycling cells and multiciliated cells undergoing centriole amplification. Both Cep57 and Cep57l1 bind to the centrosomal targeting region of Cep63. The depletion of both proteins, but not either one, blocks loading of the Cep63-Cep152 complex to mother centrioles and consequently prevents centriole duplication. We propose that Cep57 and Cep57l1 function redundantly to ensure recruitment of the Cep63-Cep152 complex to the mother centrioles for procentriole formation.

TUBright: A Peptide Probe for Imaging Microtubules.

In vitro assays using reconstituted microtubules have provided molecular insights into the principles of microtubule dynamics and the roles of microtubule-associated proteins. Emerging questions that further uncover the complexity in microtubule dynamics, especially those on tubulin isotypes and post-translational modifications, raise new technical challenges on how to visualize microtubules composed of tubulin purified from limited sources, primarily due to the low efficiency of the conventional tubulin labeling protocol. Here, we develop a peptide probe, termed TUBright, that labels in vitro reconstituted microtubules. TUBright, when coupled with different fluorescent dyes, provides flexible labeling of microtubules with a high signal-to-noise ratio. TUBright does not interfere with the dynamic behaviors of microtubules and microtubule-associated proteins. Therefore, TUBright is a useful tool for imaging microtubules, making it feasible to use tubulin from limited sources for answering many open questions on microtubule dynamics.

Correction: CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

[This corrects the article DOI: 10.1371/journal.pgen.1005485.].

Parental centrioles are dispensable for deuterosome formation and function during basal body amplification.

Mammalian epithelial cells use a pair of parental centrioles and numerous deuterosomes as platforms for efficient basal body production during multiciliogenesis. How deuterosomes form and function, however, remain controversial. They are proposed to arise either spontaneously for massive de novo centriole biogenesis or in a daughter centriole-dependent manner as shuttles to carry away procentrioles assembled at the centriole. Here, we show that both parental centrioles are dispensable for deuterosome formation. In both mouse tracheal epithelial and ependymal cells (mTECs and mEPCs), discrete deuterosomes in the cytoplasm are initially procentriole-free. They emerge at widely dispersed positions in the cytoplasm and then enlarge, concomitant with their increased ability to form procentrioles. More importantly, deuterosomes still form efficiently in mEPCs whose daughter centriole or even both parental centrioles are eliminated through shRNA-mediated depletion or drug inhibition of Plk4, a kinase essential to centriole biogenesis in both cycling cells and multiciliated cells. Therefore, deuterosomes can be assembled autonomously to mediate de novo centriole amplification in multiciliated cells.

Characterisation of centriole biogenesis during multiciliation in planarians.

BACKGROUND INFORMATION: Dense multicilia in protozoa and metazoa generate a strong force important for locomotion and extracellular fluid flow. During ciliogenesis, multiciliated cells produce hundreds of centrioles to serve as basal bodies through various pathways including deuterosome-dependent (DD), hyper-activated mother centriole-dependent (MCD) and basal bodydependent (BBD) pathways. The centrosome-free planarian Schmidtea mediterranea is widely used for regeneration studies because its neoblasts are capable of regenerating any body part after injury. However, it is currently unclear how the flatworms generate massive centrioles for multiciliated cells in the pharynx and body epidermis when their cells are initially centriole-free. RESULTS: In this study, we investigate the progress of centriole amplification during the pharynx regeneration. We observe that the planarian pharyngeal epithelial cells generate their centrioles asynchronously through a de novo pathway. Most of the de novo centrioles are formed individually, whereas the remaining ones are assembled in pairs, possibly by sharing a cartwheel, or in small clusters lacking a nucleation center. Further RNAi experiments show that the known key factors of centriole duplication, including Cep152, Plk4 and Sas6, are crucial for the centriole amplification. CONCLUSIONS AND SIGNIFICANCE: Our study demonstrates the distinct process of massive centriole biogenesis in S. mediterranea and helps to understand the diversity of centriole biogenesis during evolution.

Quadruple-layer group-IV tellurides: low thermal conductivity and high performance two-dimensional thermoelectric materials.

Through first-principles calculations, we report the thermoelectric properties of two-dimensional (2D) hexagonal group-IV tellurides XTe (X = Ge, Sn and Pb), with quadruple layers (QL) in the Te-X-X-Te stacking sequence, as promising candidates for mid-temperature thermoelectric (TE) materials. The results show that 2D PbTe exhibits a high Seebeck coefficient (∼1996 µV K-1) and a high power factor (6.10 × 1011 W K-2 m-1 s-1) at 700 K. The lattice thermal conductivities of QL GeTe, SnTe and PbTe are calculated to be 2.29, 0.29 and 0.15 W m-1 K-1 at 700 K, respectively. Using our calculated transport parameters, large values of the thermoelectric figure of merit (ZT) of 0.67, 1.90, and 2.44 can be obtained at 700 K under n-type doping for 2D GeTe, SnTe, and PbTe, respectively. Among the three compounds, 2D PbTe exhibits low average values of sound velocity (0.42 km s-1), large Grüneisen parameters (∼2.03), and strong phonon scattering. Thus, 2D PbTe shows remarkable mid-temperature TE performance with a high ZT value under both p-type (2.39) and n-type (2.44) doping. The present results may motivate further experimental efforts to verify our predictions.

Identification of amino acid residues involved in the interaction between peste-des-petits-ruminants virus haemagglutinin protein and cellular receptors.

Peste-des-petits-ruminants virus (PPRV) haemagglutinin (H) protein mediates binding to cellular receptors and then initiates virus entry. To identify the key residues of PPRV H (Hv) protein of the Nigeria 75/1 strain involved in binding to receptors, interaction of the Hv and mutated Hv (mHv) proteins with receptors (SLAM and Nectin 4) and their mutants (mSLAM1, mSLAM2, mSLAM3 and mNectin 4) was investigated using surface plasmon resonance imaging (SPRi) and coimmunoprecipitation (co-IP) assays. The results showed that the Hv protein failed to interact with mSLAM3, but interacted at a strong or medium intensity with SLAM, mSLAM2, Nectin 4 and mNectin 4, and at a low level with mSLAM1. The mHv protein was unable to interact with SLAM and its mutants, but bound to Nectin 4 and mNectin 4 with medium and weak intensity, respectively. Further analysis showed that the Hv protein could precipitate mSLAM1, mSLAM2 and mNectin 4, but not mSLAM3. The mHv protein failed to coprecipitate with SLAM and its mutants. The binding activities of mNectin 4 and Nectin 4 to mHv were less than 30.36 and 51.94 % of the wild-type levels, respectively. Based on the results obtained, amino acids at positions R389, L464, I498, R503, R533, Y541, Y543, F552 and Y553 of H protein and I61, H62, L64, K76, K78, E123, H130, I210, A211, S226 and R227 in SLAM were identified to be essential for the specificity of H-SLAM interaction, while the critical residues of H-Nectin 4 interaction require further study. These findings would improve our understanding of the invasive mechanisms of PPRV.

Rsph9 is critical for ciliary radial spoke assembly and central pair microtubule stability.

BACKGROUND INFORMATION: In the "9+2"-type motile cilia, radial spokes (RSs) protruded from the nine peripheral microtubule doublets surround and interact with the central pair (CP) apparatus to regulate ciliary beat. RSPH9 is the human homologue of the essential protozoan RS head protein Rsp9. Its mutations in human primary ciliary dyskinesia patients, however, cause CP loss in a small portion of airway cilia without affecting the ciliary localization of other head proteins. RESULTS: We characterized mouse Rsph9 and investigated its function in ependymal motile cilia. Rsph9 was specifically expressed in mouse tissues containing motile cilia and upregulated during multiciliation. Its ciliary localization complied with its putative role as an RS subunit. Depletion of Rsph9 by RNAi in mouse ependymal cilia resulted in a near complete CP loss and altered the ciliary beat pattern from planar to rotational. Multiple RS proteins, including those in the head, were also markedly downregulated in the Rsph9-depleted cilia. CONCLUSION: Rsph9 is essential for both the RS head assembly and the CP maintenance in mammalian ependymal cilia. SIGNIFICANCE: Our results help to understand the assembly and functions of mammalian RS and pathology of RS-related ciliopathy.

First-principles study of thermal transport properties in the two- and three-dimensional forms of Bi2O2Se.

Recently, an air-stable layered semiconductor Bi2O2Se has been synthesized [Nat. Nanotechnol., 2017, 12, 530; Nano Lett. 2017, 17, 3021]. It possesses ultrahigh mobility, semiconductor properties, excellent environmental stability and easy accessibility. Here, we report on the thermal transport properties in monolayer (ML), bilayer (BL), and bulk forms of Bi2O2Se using density-functional theory and the Boltzmann transport approach. The results show that the ML exhibits better thermal transport properties than the BL and bulk. The intralayer opposite phonon vibrations greatly suppress the thermal transport and lead to an ultralow lattice thermal conductivity of ∼0.74 W m-1 K-1 in the ML, which has a large band gap of ∼2.12 eV, a low value of average acoustic group velocity of ∼0.76 km s-1, low-lying optical modes of ∼0.54 THz, strong optical-acoustic phonon coupling, and large Grüneisen parameters of ∼5.69. The size effect for all three forms is much less sensitive due to their short intrinsic phonon mean free path (MFP).

First-principles calculations of thermal transport properties in MoS2/MoSe2 bilayer heterostructure.

Bilayer transition metal dichalcogenide heterostructures obtained by vertical stacking have attracted considerable attention because of their potential applications in thermoelectric and optoelectronics devices. The thermal transport behavior plays a pivotal role in assessing their functional performance. Here, we systematically investigate the thermal transport properties of the MoS2/MoSe2 bilayer heterostructure (MoS2/MoSe2-BH) by combining first-principles calculations and Boltzmann transport theory (BTE). The results show that the thermal conductivity of MoS2/MoSe2-BH at room temperature is 25.39 W m-1 K-1, which is in-between those of monolayer MoSe2 and MoS2. According to our calculated orbital-resolved phonon dispersion curves, Grüneisen parameters, phonon group velocity and relaxation time, we find that the acoustic and low-frequency optical branches below 172.65 cm-1 have strong coupling and contribute mainly to the lattice thermal conductivity. Compared with free standing monolayer MoS2 and MoSe2, the lattice thermal conductivity of MoS2/MoSe2-BH is influenced by the weak van der Waals interlayer interactions.