Hem Sapkota, Jonathan D. Wren, and Gary J. Gorbsky

Centrosomes focus microtubules to promote mitotic spindle bipolarity, a critical requirement for balanced chromosome segregation. Comprehensive understanding of centrosome function and regulation requires a complete inventory of components. While many centrosome components have been identified, others may yet remain undiscovered. We have used a bioinformatics approach, based on "guilt by association" expression to identify novel mitotic components among the large group of predicted human proteins that have yet to be functionally characterized. Here we identify Chondrosarcoma-Associated Gene 1 (CSAG1) in maintaining centrosome integrity during mitosis. Depletion of CSAG1 disrupts centrosomes and leads to multipolar spindles more effectively in cells with compromised p53 function. Thus, CSAG1 may reflect a class of "mitotic addiction" genes whose expression is more essential in transformed cells.

Aline A. Alves, Heloisa B. Gabriel, Maria J. R. Bezerra, Wanderley de Souza, Sue Vaughan, Narcisa L. Cunha-e-Silva, and Jack D. Sunter

Eukaryotic flagella are complex microtubule based organelles and in many organisms there are extra-axonemal structures present, including the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition fibre-transition zone, and the flagellum. It begins with translation of protein components, followed by their sorting and trafficking into the flagellum, transport to the assembly site and then incorporation. Flagella are formed from over 500 proteins; the principles governing axonemal component assembly are relatively clear. However, the coordination and sites of extra-axonemal structure assembly processes are less clear.

We have discovered two cytoplasmic proteins in T. brucei that are required for PFR formation, PFR assembly factors 1 and 2. Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The presence of cytoplasmic factors required for PFR formation aligns with the concept of processes occurring across multiple compartments to facilitate axoneme assembly and this is likely a common theme for extra-axonemal structure assembly.

Victor J. F. Kitano, Yoko Ohyama, Chiyomi Hayashida, Junta Ito, Mari Okayasu, Takuya Sato, Toru Ogasawara, Maki Tsujita, Akemi Kakino, Jun Shimada, Tatsuya Sawamura, and Yoshiyuki Hakeda

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO pre-OCLs, while the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR~ABCG1~PE translocation to cell surface~cell-cell fusion) in multinucleation of OCLs.

Björn Eismann, Teresa G. Krieger, Jürgen Beneke, Ruben Bulkescher, Lukas Adam, Holger Erfle, Carl Herrmann, Roland Eils, and Christian Conrad

3D cell cultures enable the in vitro study of dynamic biological processes such as the cell cycle, but their use in high-throughput screens remains impractical with conventional fluorescent microscopy. Here, we present a screening workflow for the automated evaluation of mitotic phenotypes in 3D cell cultures by light-sheet microscopy. After sample preparation by a liquid handling robot, cell spheroids are imaged for 24 hours in toto with a dual-view inverted selective plane illumination microscope (diSPIM) with a much improved signal-to-noise ratio, higher imaging speed, isotropic resolution and reduced light exposure compared to a spinning disc confocal microscope. A dedicated high-content image processing pipeline implements convolutional neural network based phenotype classification. We illustrate the potential of our approach by siRNA knock-down and epigenetic modification of 28 mitotic target genes for assessing their phenotypic role in mitosis. By rendering light-sheet microscopy operational for high-throughput screening applications, this workflow enables target gene characterization or drug candidate evaluation in tissue-like 3D cell culture models.

Su Ming Sun, Amandine Batte, Mireille Tittel-Elmer, Sophie van der Horst, Tibor van Welsem, Gordon Bean, Trey Ideker, Fred van Leeuwen, and Haico van Attikum

Eukaryotic chromosomes are replicated in interphase and the two newly duplicated sister chromatids are held together by the cohesin complex and several cohesin auxiliary factors. Sister chromatid cohesion is essential for accurate chromosome segregation during mitosis, yet has also been implicated in other processes, including DNA damage repair, transcription and DNA replication. To assess how cohesin and associated factors functionally interconnect and coordinate with other cellular processes, we systematically mapped genetic interactions of 17 cohesin genes centered on quantitative growth measurements of >52,000 gene pairs in budding yeast. Integration of synthetic genetic interactions unveiled a cohesin functional map that constitutes 373 genetic interactions, revealing novel functional connections with post-replication repair, microtubule organization and protein folding. Accordingly, we show that the microtubule-associated protein Irc15 and the prefoldin complex members Gim3, Gim4 and Yke2 are new factors involved in sister chromatid cohesion. Our genetic interaction map thus provides a unique resource for further identification and functional interrogation of cohesin proteins. Since mutations in cohesin proteins have been associated with cohesinopathies and cancer, it may also identify cohesin interactions relevant in disease etiology.

Olga Kopach, Noemi Esteras, Selina Wray, Dmitri A. Rusakov, and Andrey Y. Abramov

Frontotemporal dementia and parkinsonism (FTDP-17) caused by the 10+16 splice-site mutation in the MAPT provides an established platform to model tau-related dementia in vitro. Human iPSC-derived neurons have been shown to recapitulate the neurodevelopmental profile of tau pathology during in vitro corticogenesis as in the adult human brain. However, the neurophysiological phenotype of these cells has remained unknown, leaving unanswered questions over the functional relevance and the gnostic power of this disease model. Here we used electrophysiology to explore the membrane properties and intrinsic excitability of the generated neurons to find that human cells mature by ~150 days of neurogenesis to become compatible with matured cortical neurons. In earlier FTDP-17, neurons, however, exhibited a depolarized resting membrane potential associated with increased resistance and reduced voltage-gated Na⁺- and K⁺-channel-mediated conductance. The Na_v1.6 protein was reduced in FTDP-17. These led to a reduced cell capability of induced firing and changed action potential waveform in FTDP-17. The revealed neuropathology may thus contribute to the clinicopathological profile of the disease. This sheds new light on the significance of human models of dementia in vitro.

Tara L. Mastro, Vishnu P. Tripathi, and Susan L. Forsburg

Translesion synthesis polymerases (TLSPs) are non-essential error-prone enzymes that ensure cell survival by facilitating DNA replication in the presence of DNA damage. In addition to their role in bypassing lesions, TLSPs have been implicated in meiotic double strand break repair in several systems. Here we examine the joint contribution of four TLS polymerases to meiotic progression in the fission yeast S. pombe. We observed the dramatic loss of spore viability in fission yeast lacking all four TLSPs which is accompanied by disruptions in chromosome segregation during meiosis I and II. Rec8 cohesin dynamics are altered in the absence of the TLSPs. These data suggest that the TLSPs contribute to multiple aspects of meiotic chromosome dynamics.

Karolina Losenkova, Mariachiara Zuccarini, Marika Karikoski, Juha Laurila, Detlev Boison, Sirpa Jalkanen, and Gennady G. Yegutkin

Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects and becomes an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprised of (a) the ectoenzymatic breakdown of ATP via sequential nucleotide pyrophosphatase/phosphodiesterase-1, ecto-5’-nucleotidase/CD73 and adenosine deaminase reactions, and ATP re-synthesis through counteracting adenylate kinase and nucleoside diphosphokinase; (b) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (c) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O₂ for 24 hours triggered ~2-fold up-regulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer.

Laurence Haren, Dorian Farache, Laurent Emorine, and Andreas Merdes

-tubulin is a major protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (gamma-tubulin complex proteins): -tubulin small complexes (TuSCs), containing -tubulin and GCPs 2 and 3, and -tubulin ring complexes (TuRCs), containing multiple TuSCs, in addition to GCPs 4, 5, and 6. Whereas the structure and assembly properties of TuSCs have been intensively studied, little is known about the assembly of TuRCs, and about the specific roles of GCPs 4, 5, and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt-resistant sub-complex within the TuRC that assembles independently of the presence of TuSCs. Incubation of this sub-complex with cytoplasmic extracts containing TuSCs leads to the reconstitution of TuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the amino-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs, and we demonstrate that these are involved in the assembly or stabilization of the TuRC.

Aparna Sherlekar, Gayatri Mundhe, Prachi Richa, Bipasha Dey, Swati Sharma, and Richa Rikhy

Branched actin networks driven by Arp2/3 collaborate with actomyosin filaments in processes such as cell migration. The syncytial Drosophila blastoderm embryo also shows expansion of apical caps by Arp2/3 driven actin polymerization in interphase and buckling at contact edges by MyosinII to form furrows in metaphase. Here we study the role of Syndapin (Synd), an F-BAR domain containing protein in apical cap remodelling prior to furrow extension. synd depletion showed larger apical caps. STED super-resolution and TIRF microscopy showed long apical actin protrusions in caps in interphase and short protrusions in metaphase in control embryos. synd depletion led to sustained long protrusions even in metaphase. Loss of Arp2/3 function in synd mutants partly reverted defects in apical cap expansion and protrusion remodelling. MyosinII levels were decreased in synd mutants and MyosinII mutant embryos have been previously reported to have expanded caps. We propose that Syndapin function limits branching activity during cap expansion and affects MyosinII distribution in order to shift actin remodeling from apical cap expansion to favor lateral furrow extension.

Ana Loncar, Sergio A. Rincon, Manuel Lera Ramirez, Anne Paoletti, and Phong T. Tran

To segregate the chromosomes faithfully during cell division, cells assemble a spindle that captures the kinetochores and pulls them towards opposite poles. Proper spindle function requires correct interplay between microtubule motors and non-motor proteins. Defects in spindle assembly or changes in spindle dynamics are associated with diseases like cancer or developmental disorders. Here we compared mitotic and meiotic spindles in fission yeast. We show that even though mitotic and meiotic spindles undergo the typical three phases of spindle elongation, they have distinct features. We found that the relative concentration of kinesin-14 Pkl1 is decreased in meiosis I compared to mitosis, while the concentration of kinesin-5 Cut7 remains constant. We identified the second kinesin-14 Klp2 and microtubule dynamics as factors necessary for proper meiotic spindle assembly. This work defines differences between mitotic and meiotic spindles in fission yeast, and provides prospect for future comparative studies.

M. Pinar and M. A. Penalva

TRAnsport Protein Particle (TRAPP) complexes regulate membrane traffic. TRAPPII and TRAPPIII share a core hetero-heptamer, also denoted TRAPPI. In fungi TRAPPIII and TRAPPII mediate GDP exchange on RAB1 and RAB11, respectively, regulating traffic across the Golgi, with TRAPPIII also activating RAB1 in autophagosomes. Our finding that Aspergillus nidulans TRAPPII can be assembled by addition of a TRAPPII-specific subcomplex onto core TRAPP prompted us to investigate the possibility that TRAPPI/TRAPPIII already residing in the Golgi matures into TRAPPII to determine a RAB1-to-RAB11 conversion as Golgi cisternae progress from early Golgi to TGN identity. By time-resolved microscopy we determine that the TRAPPII reporter Trs120/TRAPPC9 is recruited to existing TGN cisternae slightly before RAB11 arrives, and resides for~45 sec on them before cisternae tear off into RAB11 secretory carriers. Notably, the core TRAPP reporter Bet3/TRAPPC3 was not detectable in early Golgi cisternae, being instead recruited to TGN cisternae simultaneously with Trs120/TRAPPC9, indicating en bloc recruitment of TRAPPII to the Golgi and arguing strongly against the TRAPP maturation model.

Javad Fahanik-babaei, Bahareh Rezaee, Maryam Nazari, Nihad Torabi, Reza Saghiri, Remy Sauve, and Afsaneh Eliassi

We have determined the electropharmacological properties of a new potassium channel from brain mitochondrial membrane by planar lipid bilayer method. Our results showed the presence of a channel with a conductance of 150 pS at potentials between 0 and –60 mV in 200 cis/50 trans mM KCl solutions.

The channel was voltage-independent, with an open probability value ~0.6 at different voltages. ATP did not affect current amplitude and Po at positive and negative voltages. Notably, adding iberiotoxin, charybdotoxin, lidocaine, and margatoxin had no effect on the channel behavior. Similarly, no changes were observed by decreasing the cis-pH to 6. Interestingly, the channel was inhibited by adding sodium in a dose dependent manner. Our results also indicated a significant increase in mitochondrial complex IV activity and membrane potential and decrease in complex I activity and mitochondrial ROS production in the presence of sodium ions.

We propose that inhibition of mitochondrial K⁺ transport by Na ions on K⁺ channel opening may be important for cell protection and ATP synthesis.

Juan Martin D'Ambrosio, Veronique Albanese, Nicolas-Frederic Lipp, Lucile Fleuriot, Delphine Debayle, Guillaume Drin, and Alenka Copic

Osh6 and Osh7 are lipid transfer proteins (LTPs) that move phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM). High PS level at the PM is key for many cellular functions. Intriguingly, Osh6/7 localize to ER-PM contact sites, although they lack membrane-targeting motifs, in contrast to multidomain LTPs that both bridge membranes and convey lipids. We show that Osh6 localization to contact sites depends on its interaction with the cytosolic tail of the ER-PM tether Ist2, a homologue of TMEM16 proteins. We identify a motif in the Ist2 tail, conserved in yeasts, as the Osh6-binding region, and we map an Ist2-binding surface on Osh6. Mutations in the Ist2 tail phenocopy osh6 osh7 deletion: they decrease cellular PS levels, and block PS transport to the PM. Our study unveils an unexpected partnership between a TMEM16-like protein and a soluble LTP, which together mediate lipid transport at contact sites.

Julian Chollet, Alexander Dünkler, Anne Bäuerle, Laura Vivero-Pol, Medhanie A. Mulaw, Thomas Gronemeyer, and Nils Johnsson

Yeast cells select the position of their new bud at the beginning of each cell cycle. The recruitment of the septins to this prospective bud site is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. Hereby, the septin-rods follow the high concentration of Cdc42_GTP that is generated by the focused localization of its GEF Cdc24. We show that shortly before budding Cdc24 not only activates Cdc42 but also transiently interacts with Cdc11, the septin subunit that caps both ends of the septin rods. Mutations in Cdc24 reducing the affinity to Cdc11 impair septin recruitment and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize into the ring, Cdc24 is found at the cortex of the bud and directs its further outgrowth from this position.

Kelly L. Dunlevy, Valentina Medvedeva, Jade E. Wilson, Mohammed Hoque, Trinity Pellegrin, Adam Maynard, Madison M. Kremp, Jason S. Wasserman, Andrey Poleshko, and Richard A. Katz

A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via "tethering proteins" that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We identified previously a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify an evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We also show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation-dephosphorylation of the LBD. Furthermore, we identified a PP2A phosphatase recognition motif within the evolutionarily conserved PRR14 C-terminal Tantalus domain. Disruption of this motif affected PRR14 localization to the nuclear lamina. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two separable, modular domains. The findings also describe an optimal PRR14 LBD fragment that could be used for efficient targeting of fusion proteins to the nuclear lamina.

Samantha-Su Z. Taylor, Nicole L. Jacobsen, Tasha K. Pontifex, Paul Langlais, and Janis M. Burt

Connexin 37 (Cx37) expression profoundly suppresses proliferation of rat insulinoma (Rin) cells in a manner dependent on gap junction channel (GJCh) functionality and the presence and phosphorylation status of its carboxyl-terminus (CT). In Rin cells growth arrested by induced Cx37 expression, serine 319 (S319) is frequently phosphorylated. Preventing phosphorylation at this site (alanine substitution; S319A) relieved Cx37 of its growth suppressive effect whereas mimicking phosphorylation at this site (aspartate substitution; S319D) enhanced Cx37's growth suppressive properties. Like Cx37-WT, -S319D GJChs and hemichannels (HChs) preferred the closed state, rarely opening fully, and gated slowly. In contrast, Cx37-S319A channels preferred open states, opened fully, and gated rapidly. These data indicate that phosphorylation-dependent conformational differences in Cx37 protein and channel function underlie Cx37-induced growth arrest vs. growth permissive phenotypes. That the closed state of -WT and Cx37-S319D GJChs and HChs favors growth arrest suggests that rather than specific permeants mediating cell cycle arrest, the closed conformation instead supports interaction of Cx37 with growth regulatory proteins that result in growth arrest.

Westley Heydeck, Brian A. Bayless, Alexander J. Stemm-Wolf, Eileen T. O'Toole, Amy S. Fabritius, Courtney Ozzello, Marina Nguyen, and Mark Winey

Basal bodies (BBs) are microtubule-based organelles that template and stabilize cilia at the cell surface. Centrins ubiquitously associate with BBs and function in BB assembly, maturation, and stability. Human POC5 (hPOC5) is a highly conserved centrin-binding protein that binds centrins through Sfi1p-like repeats and is required for building full-length, mature centrioles. Here, we use the BB-rich cytoskeleton of Tetrahymena thermophila to characterize Poc5 BB functions. Tetrahymena Poc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout of TtPOC5 leads to a significantly increased production of BBs yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A second Tetrahymena POC5-like gene, SFR1, is similarly implicated in modulating BB production. When TtPOC5 and SFR1 are co-deleted, cell viability is compromised, and levels of BB overproduction are exacerbated. Overproduced BBs display defective transition zone formation and a diminished capacity for ciliogenesis. This study uncovers a requirement for Poc5 in building mature BBs, providing a possible functional link between hPOC5 mutations and impaired cilia.

Sanjeev Chavan Nayak and Vegesna Radha

C3G (RapGEF1) plays a role in cell differentiation and is essential for early embryonic development in mice. In this study, we identify C3G as a centrosomal protein colocalizing with cenexin at the mother centriole in interphase cells. C3G interacts through its catalytic domain with cenexin, and they show interdependence for localization to the centrosome. C3G depletion caused a decrease in cellular cenexin levels. Centrosomal localization is lost as myocytes differentiate to form myotubes. Stable clone of cells depleted of C3G by CRISPR/Cas9 showed the presence of supernumerary centrioles. Overexpression of C3G, or a catalytically active deletion construct inhibited centrosome duplication. Cilia length is longer in C3G knockout cells, and the phenotype could be reverted upon reintroduction of C3G or its catalytic domain. Association of C3G with the basal body is dynamic, decreasing upon serum starvation, and increasing upon reentry into the cell cycle. C3G inhibits cilia formation and length dependent on its catalytic activity. We conclude that C3G inhibits centrosome duplication and maintains ciliary homeostasis, properties that may be important for its role in embryonic development.

Ganlu Deng, Yihong Chen, Cao Guo, Ling Yin, Ying Han, Yiyi Li, Yaojie Fu, Changjing Cai, Hong Shen, and Shan Zeng

Epithelial-mesenchymal transition (EMT) is a crucial process for cancer cells to acquire metastatic potential, which primarily causes death in gastric cancer (GC) patients. Bone morphogenetic protein 4 (BMP4) is a member of the TGF-β family that plays an indispensable role in human cancers. However, little is known about its roles in GC metastasis. In this study, BMP4 was found to be frequently overexpressed in GC tissues and was correlated with patient's poor prognosis. BMP4 was upregulated in GC cell lines and promoted EMT and metastasis of GC cells both in vitro and in vivo, while knockdown of BMP4 significantly inhibited EMT and metastasis of GC cells. Meanwhile, the inhibitor of DNA binding 1 (Id1) was identified as a downstream target of BMP4 by PCR arrays and upregulated via Smad1/5/8 phosphorylation. Id1 knockdown attenuated BMP4-induced EMT and invasion in GC cells. Moreover, Id1 overexpression in BMP4 knockdown cells restored the promotion of EMT and cell invasion. In summary, BMP4 induced EMT to promote GC metastasis by upregulating Id1 expression. Antagonizing BMP4 may be a potential therapeutic strategy in GC metastasis.

Thomas O'Loughlin, Antonina J. Kruppa, Andre L. R. Ribeiro, James R. Edgar, Abdulaziz Ghannam, Andrew M. Smith, and Folma Buss

Optineurin (OPTN) is a multifunctional protein involved in autophagy, secretion as well as NF-B and IRF3 signalling and OPTN mutations are associated with several human diseases. Here we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-B and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for ATG9A. Disease mutations linked to POAG cause aberrant foci formation in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. Using proximity labelling proteomics, we identify the LUBAC complex, CYLD and TBK1 as part of the OPTN interactome and show that these proteins are recruited to this OPTN-positive perinuclear compartment. Our work uncovers a crucial role for OPTN in dampening NF-B and IRF3 signalling through the sequestration of LUBAC and other positive regulators in this viral RNA-induced compartment leading to altered pro-inflammatory cytokine secretion.

Isabella Guardamagna, Elisabetta Bassi, Monica Savio, Paola Perucca, Ornella Cazzalini, Ennio Prosperi, and Lucia A. Stivala

Assessing DNA repair is an important endpoint to study the DNA damage response for investigating the biochemical mechanisms of this process and the efficacy of chemotherapy, which often uses DNA damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, they show some limitations mainly due to the lack of chromatin organization. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the correspondent damage-activated cytosolic extracts, containing biotin-16-dUTP, nuclei are able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 or DDB1, stimulated UVC induced dUTP incorporation. In contrast, a DDB2^PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different type of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.

Mei Wang, Luping Yu, Shu Wang, Fan Yang, Min Wang, Lufan Li, and Xin Wu

RNA-binding protein LIN28A is required for maintaining tissue homeostasis, including the reproductive system, but the underlying mechanisms on how LIN28A regulates germline progenitors remain unclear. Here, we dissected LIN28A-binding targets using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse testes. LIN28A preferentially binds to CDS or 3'UTR regions through these sites with GGAG(A) sequences enriched within mRNAs. Further investigation of Lin28a null mouse testes indicated that meiosis-associated mRNAs mediated by LIN28A were differentially expressed. Next, ribosome profiling revealed that the mRNA levels of these targets were significantly reduced in polysome fractions, and their protein expression levels decreased in the Lin28a null mouse testes, even when meiotic arrest in null mouse testes was not apparent. Collectively, these findings provide a set of binding targets that are regulated by LIN28A, which may potentially be the mechanism for the prominent role of LIN28A in regulating mammalian undifferentiated spermatogonia fates and male fertility.

Stephen Kershaw, David J. Morgan, James Boyd, David G. Spiller, Gareth Kitchen, Egor Zindy, Mudassar Iqbal, Magnus Rattray, Chris M. Sanderson, Andrew Brass, Claus Jorgensen, Tracy Hussell, Laura C. Matthews, and David W. Ray

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR) to regulate immunity, energy metabolism, and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. We show GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following glucocorticoid treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture.

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Seven corrections are made and several supplementary equations are added to the article by Yoshimura [Acta Cryst. (2015), A71, 368–381].

Structural investigations of amorphous and nanocrystalline phases forming in solution are historically challenging. Few methods are capable of in situ atomic structural analysis and rigorous control of the system. A mixed-flow reactor (MFR) is used for total X-ray scattering experiments to examine the short- and long-range structure of phases in situ with pair distribution function (PDF) analysis. The adaptable experimental setup enables data collection for a range of different system chemistries, initial supersaturations and residence times. The age of the sample during analysis is controlled by adjusting the flow rate. Faster rates allow for younger samples to be examined, but if flow is too fast not enough data are acquired to average out excess signal noise. Slower flow rates form older samples, but at very slow speeds particles settle and block flow, clogging the system. Proper background collection and subtraction is critical for data optimization. Overall, this MFR method is an ideal scheme for analyzing the in situ structures of phases that form during crystal growth in solution. As a proof of concept, high-resolution total X-ray scattering data of amorphous and crystalline calcium phosphates and amorphous calcium carbonate were collected for PDF analysis.

Appendices B4 and B5 of Cayron [Acta Cryst. (2019), A75, 411–437] contain equations involving the point group and the metric tensor in which the equality symbol should be substituted by the inclusion symbol.

Serial crystallography records still diffraction patterns from single, randomly oriented crystals, then merges data from hundreds or thousands of them to form a complete data set. To process the data, the diffraction patterns must first be indexed, equivalent to determining the orientation of each crystal. A novel automatic indexing algorithm is presented, which in tests usually gives significantly higher indexing rates than alternative programs currently available for this task. The algorithm does not require prior knowledge of the lattice parameters but can make use of that information if provided, and also allows indexing of diffraction patterns generated by several crystals in the beam. Cases with a small number of Bragg spots per pattern appear to particularly benefit from the new approach. The algorithm has been implemented and optimized for fast execution, making it suitable for real-time feedback during serial crystallography experiments. It is implemented in an open-source C++ library and distributed under the LGPLv3 licence. An interface to it has been added to the CrystFEL software suite.

Single-wavelength X-ray anomalous diffraction (SAD) is a frequently employed technique to solve the phase problem in X-ray crystallography. The precision and accuracy of recovered anomalous differences are crucial for determining the correct phases. Continuous rotation (CR) and inverse-beam geometry (IBG) anomalous data collection methods have been performed on tetragonal lysozyme and monoclinic survivin crystals and analysis carried out of how correlated the pairs of Friedel's reflections are after scaling. A multivariate Bayesian model for estimating anomalous differences was tested, which takes into account the correlation between pairs of intensity observations and incorporates the a priori knowledge about the positivity of intensity. The CR and IBG data collection methods resulted in positive correlation between I(+) and I(−) observations, indicating that the anomalous difference dominates between these observations, rather than different levels of radiation damage. An alternative pairing method based on near simultaneously observed Bijvoet's pairs displayed lower correlation and it was unsuccessful for recovering useful anomalous differences when using the multivariate Bayesian model. In contrast, multivariate Bayesian treatment of Friedel's pairs improved the initial phasing of the two tested crystal systems and the two data collection methods.

Under the anomalous transmission condition in the Bragg mode, X-ray interference fringes were observed between two beams with different hyperbolic trajectories in a very weakly bent plane-parallel perfect crystal with negative strain gradient. The origin of the fringes was analysed based on the dynamical theory of diffraction for a distorted crystal. In the reflected beam from the entrance surface, the interference fringes were observed between once- and twice-reflected beams from the back surface. In the transmitted beam from the back surface, the interference fringes were observed between the direct beam and once-reflected beam from the entrance surface. In the emitted beam from the lateral surface, the interference fringes were observed between the beams after different numbers of reflections in the crystal. The multiply reflected beams were formed by a combined result of long propagation length along the beam direction with large divergence of the refracted beams when the strain gradient was negative. The period of these interference fringes was sensitive to very weak strain, of the order of 10−7.

Relativistic electron diffraction depends on linear and quadratic terms in the electric potential, the latter being neglected in the frequently used relativistically corrected Schrödinger equation. The quadratic electric potential term modifies atomic scattering amplitudes in particular for large-angle scattering and backscattering. The respective correction increases with increasing scattering angle, increasing atomic number and increasing kinetic energy. Conventional tabulations for electron scattering and its large-angle extrapolations can be amended in closed form by a universal correction based on the screened Coulomb potential squared.

This work studies the elastic scattering behavior of electron vortices when propagating through amorphous samples. A formulation of the multislice approach in cylindrical coordinates is used to theoretically investigate the redistribution of intensity between different angular momentum components due to scattering. To corroborate and elaborate on our theoretical results, extensive numerical simulations are performed on three model systems (Si3N4, Fe0.8B0.2, Pt) for a wide variety of experimental parameters to quantify the purity of the vortices, the net angular momentum transfer, and the variability of the results with respect to the random relative position between the electron beam and the scattering atoms. These results will help scientists to further improve the creation of electron vortices and enhance applications involving them.

A novel approach for finding and evaluating structural models of small metallic nanoparticles is presented. Rather than fitting a single model with many degrees of freedom, libraries of clusters from multiple structural motifs are built algorithmically and individually refined against experimental pair distribution functions. Each cluster fit is highly constrained. The approach, called cluster-mining, returns all candidate structure models that are consistent with the data as measured by a goodness of fit. It is highly automated, easy to use, and yields models that are more physically realistic and result in better agreement to the data than models based on cubic close-packed crystallographic cores, often reported in the literature for metallic nanoparticles.

The use of strongly bent crystals in spectrometers for pulses of a hard X-ray free-electron laser is explored theoretically. Diffraction is calculated in both dynamical and kinematical theories. It is shown that diffraction can be treated kinematically when the bending radius is small compared with the critical radius given by the ratio of the Bragg-case extinction length for the actual reflection to the Darwin width of this reflection. As a result, the spectral resolution is limited by the crystal thickness, rather than the extinction length, and can become better than the resolution of a planar dynamically diffracting crystal. As an example, it is demonstrated that spectra of the 12 keV pulses can be resolved in the 440 reflection from a 20 µm-thick diamond crystal bent to a radius of 10 cm.

The transformations from the primitive cells of the centered Bravais lattices to the corresponding centered cells have conventionally been listed as three-by-three matrices that transform three-space lattice vectors. Using those three-by-three matrices when working in the six-dimensional space of lattices represented as Selling scalars as used in Delone (Delaunay) reduction, one could transform to the three-space representation, apply the three-by-three matrices and then back-transform to the six-space representation, but it is much simpler to have the equivalent six-by-six matrices and apply them directly. The general form of the transformation from the three-space matrix to the corresponding matrix operating on Selling scalars (expressed in space S6) is derived, and the particular S6matrices for the centered Delone types are listed. (Note: in his later publications, Boris Delaunay used the Russian version of his surname, Delone.)

A crystallographic indexing algorithm, pinkIndexer, is presented for the analysis of snapshot diffraction patterns. It can be used in a variety of contexts including measurements made with a monochromatic radiation source, a polychromatic source or with radiation of very short wavelength. As such, the algorithm is particularly suited to automated data processing for two emerging measurement techniques for macromolecular structure determination: serial pink-beam X-ray crystallography and serial electron crystallography, which until now lacked reliable programs for analyzing many individual diffraction patterns from crystals of uncorrelated orientation. The algorithm requires approximate knowledge of the unit-cell parameters of the crystal, but not the wavelengths associated with each Bragg spot. The use of pinkIndexer is demonstrated by obtaining 1005 lattices from a published pink-beam serial crystallography data set that had previously yielded 140 indexed lattices. Additionally, in tests on experimental serial crystallography diffraction data recorded with quasi-monochromatic X-rays and with electrons the algorithm indexed more patterns than other programs tested.

X-ray emission under electron-channelling conditions is used to distinguish between a non-centrosymmetric half-Heusler and a centrosymmetric full-Heusler crystal. For TiCo1.5+xSn the space-group determination based on a Rietveld refinement procedure became challenging for increasing Co content (x > 0.2), while electron channelling proved successful for higher Co content (x = 0.35). This technique can be used on crystals as small as (10 nm)3.

In this study, the atomic structure of the ternary icosahedral ZnMgTm quasicrystal (QC) is investigated by means of single-crystal X-ray diffraction. The structure is found to be a member of the Bergman QC family, frequently found in Zn–Mg–rare-earth systems. The ab initio structure solution was obtained by the use of the Superflip software. The infinite structure model was founded on the atomic decoration of two golden rhombohedra, with an edge length of 21.7 Å, constituting the Ammann–Kramer–Neri tiling. The refined structure converged well with the experimental diffraction diagram, with the crystallographic R factor equal to 9.8%. The Bergman clusters were found to be bonded by four possible linkages. Only two linkages, b and c, are detected in approximant crystals and are employed to model the icosahedral QCs in the cluster approach known for the CdYb Tsai-type QC. Additional short b and a linkages are found in this study. Short interatomic distances are not generated by those linkages due to the systematic absence of atoms and the formation of split atomic positions. The presence of four linkages allows the structure to be pictured as a complete covering by rhombic triacontahedral clusters and consequently there is no need to define the interstitial part of the structure (i.e. that outside the cluster). The 6D embedding of the solved structure is discussed for the final verification of the model.

Entangled embedded periodic nets and crystal frameworks are defined, along with their dimension type, homogeneity type, adjacency depth and periodic isotopy type. Periodic isotopy classifications are obtained for various families of embedded nets with small quotient graphs. The 25 periodic isotopy classes of depth-1 embedded nets with a single-vertex quotient graph are enumerated. Additionally, a classification is given of embeddings of n-fold copies of pcu with all connected components in a parallel orientation and n vertices in a repeat unit, as well as demonstrations of their maximal symmetry periodic isotopes. The methodology of linear graph knots on the flat 3-torus [0,1)3 is introduced. These graph knots, with linear edges, are spatial embeddings of the labelled quotient graphs of an embedded net which are associated with its periodicity bases.

Crystal structure identification of thin organic films entails a number of technical and methodological challenges. In particular, if molecular crystals are epitaxially grown on single-crystalline substrates a complex scenario of multiple preferred orientations of the adsorbate, several symmetry-related in-plane alignments and the occurrence of unknown polymorphs is frequently observed. In theory, the parameters of the reduced unit cell and its orientation can simply be obtained from the matrix of three linearly independent reciprocal-space vectors. However, if the sample exhibits unit cells in various orientations and/or with different lattice parameters, it is necessary to assign all experimentally obtained reflections to their associated individual origin. In the present work, an effective algorithm is described to accomplish this task in order to determine the unit-cell parameters of complex systems comprising different orientations and polymorphs. This method is applied to a polycrystalline thin film of the conjugated organic material 6,13-pentacene­quinone (PQ) epitaxially grown on an Ag(111) surface. All reciprocal vectors can be allocated to unit cells of the same lattice constants but grown in various orientations [sixfold rotational symmetry for the contact planes (102) and (102)]. The as-determined unit cell is identical to that reported in a previous study determined for a fibre-textured PQ film. Preliminary results further indicate that the algorithm is especially effective in analysing epitaxially grown crystallites not only for various orientations, but also if different polymorphs are present in the film.

Recent developments of two-color operation modes at X-ray free-electron laser facilities provide new research opportunities, such as X-ray pump/X-ray probe experiments and multiple-wavelength anomalous dispersion phasing methods. However, most existing indexing methods were developed for indexing diffraction data from monochromatic X-ray beams. Here, a new algorithm is presented for indexing two-color diffraction data, as an extension of the sparse-pattern indexing algorithm SPIND, which has been demonstrated to be capable of indexing diffraction patterns with as few as five peaks. The principle and implementation of the two-color indexing method, SPIND-TC, are reported in this paper. The algorithm was tested on both simulated and experimental data of protein crystals. The results show that the diffraction data can be accurately indexed in both cases. Source codes are publicly available at https://github.com/lixx11/SPIND-TC.

A new approach is presented to obtain candidate structures from atomic pair distribution function (PDF) data in a highly automated way. It fetches, from web-based structural databases, all the structures meeting the experimenter's search criteria and performs structure refinements on them without human intervention. It supports both X-ray and neutron PDFs. Tests on various material systems show the effectiveness and robustness of the algorithm in finding the correct atomic crystal structure. It works on crystalline and nanocrystalline materials including complex oxide nanoparticles and nanowires, low-symmetry and locally distorted structures, and complicated doped and magnetic materials. This approach could greatly reduce the traditional structure searching work and enable the possibility of high-throughput real-time auto-analysis PDF experiments in the future.

The generalized Darwin–Hamilton equations [Wuttke (2014). Acta Cryst. A70, 429–440] describe multiple Bragg reflection from a thick, ideally imperfect crystal. These equations are simplified by making full use of energy conservation, and it is demonstrated that the conventional two-ray Darwin–Hamilton equations are obtained as a first-order approximation. Then an efficient numeric solution method is presented, based on a transfer matrix for discretized directional distribution functions and on spectral collocation in the depth coordinate. Example solutions illustrate the orientational spread of multiply reflected rays and the distortion of rocking curves, especially if the detector only covers a finite solid angle.