Peripheral astral microtubules ensure asymmetric furrow positioning in neural stem cells.

Neuroblast division is characterized by asymmetric positioning of the cleavage furrow, resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly are governed by centralspindlin that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In neuroblasts, these two centralspindlin populations are spatially and temporally separated. A leading pool is located at the basal cleavage site and a second pool accumulates at the midzone before traveling to the cleavage site. The cortical centralspindlin population requires peripheral astral microtubules and the chromosome passenger complex for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin signaling at the midzone, leading to equatorial furrow repositioning and decreased size asymmetry. These data show that basal furrow positioning in neuroblasts results from a competition between different centralspindlin pools in which the cortical pool is dominant.

Chitin Deacetylases Are Required for Epichloë festucae Endophytic Cell Wall Remodeling During Establishment of a Mutualistic Symbiotic Interaction with Lolium perenne.

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network and also grows as an epiphyte. However, little is known about the cell wall-remodeling mechanisms required to avoid host defense and maintain intercalary growth within the host. Here, we use a suite of molecular probes to show that the E. festucae cell wall is remodeled by conversion of chitin to chitosan during infection of L. perenne seedlings, as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth, the cell wall is remodeled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalyzed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption of fungal intercalary growth in the intercellular spaces of plants infected with these mutants. These results establish that these two genes are required for maintenance of the mutualistic symbiotic interaction between E. festucae and L. perenne.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Campylobacter novaezeelandiae sp. nov., isolated from birds and water in New Zealand.

Six isolates of Campylobacter with similar non-standard colonial morphologies were identified during studies isolating Campylobacter from bird faeces and rivers in New Zealand. Genomic (16S rRNA gene sequencing and whole genome analysis) and phenotypic (MALDI-TOF analysis and conventional biochemical tests) showed that the isolates form a monophyletic clade with genetic relationships to Campylobacter coli/Campylobacter jejuni and Campylobacter peloridis/Campylobacter amoricus. They may be distinguished from other Campylobacter by their MALDI-TOF spectral pattern, their florid α-haemolysis, their ability to grow anaerobically at 37 °C, and on 2 % NaCl nutrient agar, and their lack of hippuricase. This study shows that these isolates represent a novel species within the genus Campylobacter for which the name Campylobacter novaezeelandiae sp. nov. is proposed. The presence of C. novaezeelandiae in water may be a confounder for freshwater microbial risk assessment as they may not be pathogenic for humans. The type strain is B423bT (=NZRM 4741T=ATCC TSD-167T).

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis.

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyze the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analyzed by light-, confocal- and electron microscopy. Changes in PA distribution were analyzed in E. festucae using a PA biosensor and the impact of these changes on the endocytic recycling and superoxide production investigated. We found that E. festucae PldB, and the N. crassa ortholog, PLA-7, are required for polarized growth and cell fusion and contribute to ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network for hyphal morphogenesis and growth of filamentous fungi.

A homologue of the fungal tetraspanin Pls1 is required for Epichloë festucae expressorium formation and establishment of a mutualistic interaction with Lolium perenne.

Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with the grass host Lolium perenne. Endophytic hyphae exit the host by an appressorium-like structure known as an expressorium. In plant-pathogenic fungi, the tetraspanin Pls1 and the NADPH oxidase component Nox2 are required for appressorium development. Previously we showed that the homologue of Nox2, NoxB, is required for E. festucae expressorium development and establishment of a mutualistic symbiotic interaction with the grass host. Here we used a reverse genetics approach to functionally characterize the role of the E. festucae homologue of Pls1, PlsA. The morphology and growth of ΔplsA in axenic culture was comparable to wild-type. The tiller length of plants infected with ΔplsA was significantly reduced. Hyphae of ΔplsA had a proliferative pattern of growth within the leaves of L. perenne with increased colonization of the intercellular spaces and the vascular bundles. The ΔplsA mutant was also defective in expressorium development although the phenotype was not as severe as for ΔnoxB, highlighting potentially distinct roles for PlsA and NoxB in signalling through the NoxB complex. Hyphae of ΔplsA proliferate below the cuticle surface but still occasionally form an expressorium-like structure that enables the mutant hyphae to exit the leaf to grow on the surface. These expressoria still form a septin ring-like structure at the point of cuticle exit as found in the wild-type strain. These results establish that E. festucae PlsA has an important, but distinct, role to NoxB in expressorium development and plant symbiosis.

Genomic correlates of extraintestinal infection are linked with changes in cell morphology in Campylobacter jejuni.

Campylobacter jejuni is the most common cause of bacterial diarrheal disease in the world. Clinical outcomes of infection can range from asymptomatic infection to life-threatening extraintestinal infections. This variability in outcomes for infected patients has raised questions as to whether genetic differences between C. jejuni isolates contribute to their likelihood of causing severe disease. In this study, we compare the genomes of ten C. jejuni isolates that were implicated in extraintestinal infections with reference gastrointestinal isolates, in order to identify unusual patterns of sequence variation associated with infection outcome. We identified a collection of genes that display a higher burden of uncommon mutations in invasive isolates compared with gastrointestinal close relatives, including some that have been previously linked to virulence and invasiveness in C. jejuni. Among the top genes identified were mreB and pgp1, which are both involved in determining cell shape. Electron microscopy confirmed morphological differences in isolates carrying unusual sequence variants of these genes, indicating a possible relationship between extraintestinal infection and changes in cell morphology.

Abomasal dysfunction and cellular and mucin changes during infection of sheep with larval or adult Teladorsagia circumcincta.

This is the first integrated study of the effects on gastric secretion, inflammation and fundic mucins after infection with L3 T. circumcincta and in the very early period following transplantation of adult worms. At 3 months-of-age, 20 Coopworth lambs were infected intraruminally with 35,000 L3; infected animals were killed on Days 5, 10, 15, 20 and 30 post-infection and 6 controls on either Day 0 or 30 post-infection. Another 15 Romney cross lambs received 10,000 adult worms at 4-5 months-of-age though surgically-implanted abomasal cannulae and were killed after 6, 12, 24 and 72 hours; uninfected controls were also killed at 72 hours. Blood was collected at regular intervals from all animals for measurement of serum gastrin and pepsinogen and abomasal fluid for pH measurement from cannulated sheep. Tissues collected at necropsy were fixed in Bouin's fluid for light microscopy, immunocytochemistry and mucin staining and in Karnovsky's fluid for electron microscopy. Nodules around glands containing developing larvae were seen on Day 5 p.i., but generalised effects on secretion occurred only after parasite emergence and within hours after transplantation of adult worms. After L3 infection, there were maximum worm burdens on Days 10-15 post-infection, together with peak tissue eosinophilia, inhibition of gastric acid secretion, hypergastrinaemia, hyperpepsinogenaemia, loss of parietal cells, enlarged gastric pits containing less mucin and increased numbers of mucous neck cells. After adult transplantation, serum pepsinogen was significantly increased after 9 hours and serum gastrin after 18 hours. Parallel changes in host tissues and the numbers of parasites in the abomasal lumen suggest that luminal parasites, but not those in the tissues, are key drivers of the pathophysiology and inflammatory response in animals exposed to parasites for the first time. These results are consistent with initiation of the host response by parasite chemicals diffusing across the surface epithelium, possibly aided by components of ES products which increased permeability. Parietal cells appear to be a key target, resulting in secondary increases in serum gastrin, pit elongation, loss of surface mucins and inhibition of chief cell maturation. Inflammation occurs in parallel, and could either cause the pathology or exacerbate the direct effects of ES products.

Microscopy Methods for Analysis of Spindle Dynamics in Meiotic Drosophila Spermatocytes.

The spindle is a microtubule-based structure whose remodeling is required for partitioning the chromosomes and cytoplasm during meiosis. Characterizing microtubule behavior is fundamental to understanding how these tubulin polymers contribute to successful cell division. Here, a procedure is described for the imaging and analysis of spindle microtubule dynamics in cultures of living Drosophila melanogaster primary spermatocytes expressing tubulin tagged with enhanced green fluorescent protein. It employs time-lapse scanning confocal microscopy and the photobleaching of fiduciary marks onto fluorescently tagged microtubules. These labels are subsequently used to determine the sites and rates of kinetochore fiber growth and shrinkage during metaphase. This method can be readily applied to different microtubule populations, meiotic stages, and genetic backgrounds.

Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

BACKGROUND: Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. RESULTS: Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. CONCLUSION: Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes.

In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems.

Centromeric binding and activity of Protein Phosphatase 4.

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis.

Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila.

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis.

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.

Drosophila Klp67A binds prophase kinetochores to subsequently regulate congression and spindle length.

The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite their highly conserved roles in chromosome alignment and spindle dynamics, remain poorly characterised. Here, we report that the Drosophila kinesin-8 protein, Klp67A, exists in two spatially and functionally separable metaphase pools: at kinetochores and along the spindle. Fixed and live-cell analyses of different Klp67A recombinant variants indicate that this kinesin-8 first collects at kinetochores during prophase and, by metaphase, localises to the kinetochore outerplate. Although the catalytic motor activity of Klp67A is required for efficient kinetochore recruitment at all times, microtubules are entirely dispensable for this process. The tail of Klp67A does not play a role in kinetochore accumulation, but is both necessary and sufficient for spindle association. Using functional assays, we reveal that chromosome position and spindle length are determined by the microtubule-depolymerising motor activity of Klp67A exclusively when located at kinetochores, but not along the spindle. These data reveal that, unlike other metazoan kinesin-8 proteins, Klp67A binds the nascent prophase and mature metaphase kinetochore. From this location, Klp67A uses its motor activity to ensure chromosome alignment and proper spindle length.

Girds 'n' cleeks o' cytokinesis: microtubule sticks and contractile hoops in cell division.

Microtubules maintain an intimate relationship with the rings of anillin, septins and actomyosin filaments throughout cytokinesis. In Drosophila, peripheral microtubules emanating from the spindle poles contact the equatorial cell cortex to deliver the signal that initiates formation of the cytokinetic furrow. Mutations that affect microtubule stability lead to ectopic furrowing because peripheral microtubules contact inappropriate cortical sites. The PAV-KLP (Pavarotti-kinesin-like protein)/RacGAP50C (where GAP is GTPase-activating protein) centralspindlin complex moves towards the plus ends of microtubules to reach the cell equator. When RacGAP50C is tethered to the cell membrane, furrowing initiates at multiple non-equatorial sites, indicating that mis-localization of this single molecule is sufficient to promote furrowing. Furrow formation and ingression requires RhoA activation by the RhoGEF (guanine-nucleotide-exchange factor) Pebble, which interacts with RacGAP50C. RacGAP50C also binds anillin, which associates with actin, myosin and septins. Thus RacGAP50C plays a pivotal role during furrow formation by activating RhoA and linking the peripheral microtubules with the nascent rings through its interaction with anillin.

RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis.

Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, time-lapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAP-signaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site.

Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo.

The metaphase-spindle steady-state length occurs as spindle microtubules ;flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance.

The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation.

The CDK11 (cyclin-dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110-kDa isoform is expressed at constant levels during the cell cycle and the shorter 58-kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. gamma-Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11(p58) isoform, but not the CDK11(p110) isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11(p58) in centrosome maturation and bipolar spindle morphogenesis.

Klp67A destabilises pre-anaphase microtubules but subsequently is required to stabilise the central spindle.

Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which results in abnormally long and stable pre-anaphase microtubules. Here we examine its role during cytokinesis in Drosophila primary spermatocytes that require the coordinated interaction of an interior and peripheral set of central spindle microtubules. In mutants anaphase B spindles elongated with normal kinetics but bent towards the cortex. Both peripheral and interior spindle microtubules then formed diminished bundles of abnormally positioned central spindle microtubules associated with the pavarotti-KLP and KLP3A motor proteins. The minus ends of these were poorly aligned as revealed by Asp protein localisation. Furrows always initiated at the sites of central spindle bundles but could be unilateral or nonequatorially positioned. Ectopic furrows were stimulated by the interior central spindle and formed only after this structure buckled and contacted the cortex. Furrows often halted and regressed as they could not be sustained by the central spindles that became increasing unstable over time and often completely degraded. Consistent with this, actin and anillin failed to form homogenous bands. Thus, the Klp67A microtubule catastrophe factor is required for cytokinesis by regulating both the formation and stability of the central spindle.

Cleavage furrow formation and ingression during animal cytokinesis: a microtubule legacy.

Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independent daughter cells at the end of cell division. Although the metazoan mitotic spindle has been implicated in the placement and advancement of the cleavage furrow, the molecules responsible for these processes have remained elusive. Recent studies have provided insights into the role of different microtubule structures and associated proteins in cleavage furrow positioning and ingression together with the signalling events that regulate the dynamics of the equatorial cell cortex during cytokinesis. We try to unify these findings into a general model of cytokinesis in which both astral and central spindle microtubules have the ability to induce furrowing. We further propose that the evolutionarily conserved centralspindlin complex serves as a master controller of cell cleavage in Drosophila by promoting both furrow formation and ingression. The same mechanism might be conserved in other organisms.