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Ocean microbial communities strongly influence the biogeochemistry, food webs, and climate of our planet. Despite recent advances in understanding their taxonomic and genomic compositions, little is known about how their transcriptomes vary globally. Here, we present a dataset of 187 metatranscriptomes and 370 metagenomes from 126 globally distributed sampling stations and establish a resource of 47 million genes to study community-level transcriptomes across depth layers from pole-to-pole. We examine gene expression changes and community turnover as the underlying mechanisms shaping community transcriptomes along these axes of environmental variation and show how their individual contributions differ for multiple biogeochemically relevant processes. Furthermore, we find the relative contribution of gene expression changes to be significantly lower in polar than in non-polar waters and hypothesize that in polar regions, alterations in community activity in response to ocean warming will be driven more strongly by changes in organismal composition than by gene regulatory mechanisms. VIDEO ABSTRACT., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

An Aurora A regulatory module has been identified in two different proteins: TPX2 in Xenopus laevis and TPXL-1 in C. elegans. The diverse roles of these two proteins in spindle assembly leave us to beckon the true C. elegans TPX2 ortholog to center stage.

In higher eukaryotes, microtubules (MT) in both halves of the mitotic spindle translocate continuously away from the midzone in a phenomenon called poleward microtubule flux. Because the spindle maintains constant length and microtubule density, this microtubule translocation must somehow be coupled to net MT depolymerization at spindle poles. The molecular mechanisms underlying both flux-associated translocation and flux-associated depolymerization are not well understood, but it can be predicted that blocking pole-based destabilization will increase spindle length, an idea that has not been tested in meiotic spindles. Here, we show that simultaneous addition of two pole-disrupting reagents p50/dynamitin and a truncated version of Xklp2 results in continuous spindle elongation in Xenopus egg extracts, and we quantitatively correlate this elongation rate with the poleward translocation of stabilized microtubules. We further use this system to demonstrate that this poleward translocation requires the activity of the kinesin-related protein Eg5. These results suggest that Eg5 is responsible for flux-associated MT translocation and that dynein and Xklp2 regulate flux-associated microtubule depolymerization at spindle poles.

The mitotic spindle of animal cells is a bipolar array of microtubules that guides chromosome segregation during cell division. It has been proposed that during spindle assembly chromatin can positively influence microtubule stability at a distance from its surface throughout its neighboring cytoplasm. However, such an "à distance" effect has never been visualized directly. Here, we have used centrosomal microtubules and chromatin beads to probe the regulation of microtubule behavior around chromatin in Xenopus egg extracts. We show that, in this system, chromatin does affect microtubule formation at a distance, inducing preferential orientation of centrosomal microtubules in its direction. Moreover, this asymmetric distribution of microtubules is translated into a directional migration of centrosomal asters toward chromatin and their steady-state repositioning within 10 microm of chromatin. To our knowledge, this is the first direct evidence of a long-range guidance effect at the sub-cellular level.

Microtubules are essential structures that organize the cytoplasm and form the mitotic spindle. Their number and orientation depend on the rate of nucleation events and their dynamics. Microtubules are often, but not always, nucleated off a single cytoplasmic element, the centrosome. One microtubule-associated protein, XMAP215, is also a resident centrosomal protein. In this study, we have found that XMAP215 is a key component for the microtubule-nucleating activity of centrosomes. We show that depletion of XMAP215 from Xenopus egg extracts impairs their ability to reconstitute the microtubule nucleation potential of salt-stripped centrosomes. We also show that XMAP215 immobilized on polymer beads induces the formation of microtubule asters in egg extracts as well as in solutions of pure tubulin. Formation of asters by XMAP215 beads indicates that this protein is able to anchor nascent microtubules via their minus ends. The aster-forming activity of XMAP215 does not require gamma-tubulin in pure tubulin solutions, but it is gamma-tubulin-dependent in egg extracts. Our results indicate that XMAP215, a resident centrosomal protein, contributes to the microtubule-nucleating activity of centrosomes, suggesting that, in vivo, the formation of asters by centrosomes requires factors additional to gamma-tubulin.

The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin alpha, and is displaced from importin alpha by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin alpha. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase.

Metaphase chromosome alignment is a key step of animal cell mitosis. The molecular mechanism leading to this equatorial positioning is still not fully understood. Forces exerted at kinetochores and on chromosome arms drive chromosome movements that culminate in their alignment on the metaphase plate. In this paper, we show that Xkid, a kinesin-like protein localized on chromosome arms, plays an essential role in metaphase chromosome alignment and in its maintenance. We propose that Xkid is responsible for the polar ejection forces acting on chromosome arms. Our results show that these forces are essential to ensure that kinetochores and chromosome arms align on a narrow equatorial plate during metaphase, a prerequisite for proper chromosome segregation.

Background: In eukaryotes, assembly of the mitotic spindle requires the interaction of chromosomes with microtubules. During this process, several motor proteins that move along microtubules promote formation of a bipolar microtubule array, but the precise mechanism is unclear. In order to examine the roles of different motor proteins in building a bipolar spindle, we have used a simplified system in which spindles assemble around beads coated with plasmid DNA and incubated in extracts from Xenopus eggs. Using this system, we can study spindle assembly in the absence of paired cues, such as centrosomes and kinetochores, whose microtubule-organizing properties might mask the action of motor proteins., Results: We blocked the function of individual motor proteins in the Xenopus extracts using specific antibodies. Inhibition of Xenopus kinesin-like protein 1 (Xklp1) led either to the dissociation of chromatin beads from microtubule arrays, or to collapsed microtubule bundles on beads. Inhibition of Eg5 resulted in monopolar microtubule arrays emanating from chromatin beads. Addition of antibodies against dynein inhibited the focusing of microtubule ends into spindle poles in a dose-dependent manner. Inhibition of Xenopus carboxy-terminal kinesin 2 (XCTK2) affected both pole formation and spindle stability. Co-inhibition of XCTK2 and dynein dramatically increased the severity of spindle pole defects. Inhibition of Xklp2 caused only minor spindle pole defects., Conclusions: Multiple microtubule-based motor activities are required for the bipolar organization of microtubules around chromatin beads, and we propose a model for the roles of the individual motor proteins in this process.

Microtubules are implicated in the movement and positioning of nuclei in many cell types. Nuclei can be moved by forces acting on microtubules nucleated at the spindle pole body, as in fungi [1], or microtubules nucleated at the centrosome, as during migration of the male (sperm) pronucleus towards the centre of the zygote after fertilization [2] [3] [4]. The dramatic movements of the female pronucleus towards the male pronucleus potentially involve another mechanism: movement along the microtubules of the sperm aster towards their slower growing, attached or 'minus' ends [3] [5]. Here, we have reconstituted this last type of nuclear movement in vitro. Synthetic nuclei assembled in cytoplasmic extracts made from interphase Xenopus eggs move along microtubules towards their minus ends. We provide strong experimental evidence that cytoplasmic dynein is the motor for nuclear movement in this in vitro system, and discuss our results in terms of current knowledge of motility of the endoplasmic reticulum.

We describe a novel Xenopus plus end-directed kinesin-like protein (KLP), Xklp2, localized on centrosomes throughout the cell cycle and on spindle pole microtubules during metaphase. Using mitotic spindles assembled in Xenopus egg extracts and different recombinant GST-Xklp2 mutants, we show that this motor is targeted to spindle poles through its C-terminal domain. Xklp2-truncated polypeptides lacking the motor domain block centrosome separation and disrupt preassembled metaphase spindles. Antibodies directed against the tail of Xklp2 have a similar effect. These results show that Xklp2 protein is required for centrosome separation and maintenance of spindle bipolarity. This study is an example of the application of the dominant negative mutant effect on spindle assembly in Xenopus egg extracts, demonstrating the usefulness of this approach in probing the function of proteins in this system.

Background: The microtubule network, upon which transport occurs in higher cells, is formed by the polymerization of alpha and beta tubulin. The third major tubulin isoform, gamma tubulin, is believed to serve a role in organizing this network by nucleating microtubule growth on microtubule-organizing centers, such as the centrosome. Research in vitro has shown that gamma tubulin must be restored to stripped centrioles to regenerate the centrosomal functions of duplication and microtubule nucleation., Results: We have re-examined the localization of gamma tubulin in isolated and in situ mammalian centrosomes using a novel immunocytochemical technique that preserves antigenicity and morphology while allowing increased accessibility. As expected, alpha tubulin was localized in cytoplasmic and centriolar barrel microtubules and in the associated pericentriolar material. Foci of gamma tubulin were observed at the periphery of the organized pericentriolar material, as reported previously, often near the termini of microtubules. A further and major location of gamma tubulin was a structure within the proximal end of the centriolar barrel. The distributions were complementary, in that alpha tubulin was excluded from the core of the centriole, and gamma tubulin was excluded from the microtubule barrel., Conclusions: We have shown that gamma tubulin is localized both in the pericentriolar material and in the core of the mammalian centriole. This result suggests that gamma tubulin has a role in the centriolar duplication process, perhaps as a template for growth of the centriolar microtubules, in addition to its established role in the nucleation of astral microtubules.

Xklp1 is a novel Xenopus kinesin-like protein with a motor domain at the amino terminus, nuclear localization sequences in the stalk, and a putative zinc finger-like sequence in the tail. It is nuclear during interphase and chromosomal during mitosis. During late anaphase, a fraction of the protein relocalizes to the spindle interzone and accumulates in the midbody during telophase. Depletion of Xklp1 protein by antisense oligo knockout in oocytes leads to defective mitosis during the first cell cycles following fertilization. The bipolarity of spindles assembled in vitro in the presence of anti-Xklp1 antibodies is unstable, and the chromosomes fail to congress on the metaphase plate.

Activated Xenopus eggs show periodic surface contraction waves and oscillations in endogenous protein phosphorylation, MPF, and kinase activities timed with the cleavage cycle of control fertilized eggs. In this paper, we show that in activated eggs lacking the material that originates from the oocyte nucleus, MPF and kinase oscillations occur in the absence of surface contraction waves. Two mitotic phosphoproteins (M116 and M46), previously described by 32P labeling in nucleated eggs, are no longer detected in the enucleated eggs. We conclude that a cytoplasmic temporal control of MPF and kinase activities is likely to be the essential cell cycle oscillator. The oocyte nuclear components normally stored in the cytoplasm of the embryos are not involved in the clock although they appear to be required for the generation of surface contraction waves.