More than Two Populations of Microtubules Comprise the Dynamic Mitotic Spindle

The microtubules of the mitotic spindle mediate chromosome alignment to the metaphase plate, then sister chromatid segregation to the spindle poles in anaphase. Previous analyses of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, and these were ascribed to reflect kinetochore versus non-kinetochore microtubules, respectively. Here, we test this categorization by disrupting kinetochores through depletion of the Ndc80 complex. In the absence of functional kinetochores, microtubule dynamics still exhibit slow and fast turnover populations, though the proportion of each population and the timings of turnover are altered. Importantly, the data obtained following Hec1/Ndc80 depletion suggests other sub-populations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. For example, while Aurora B kinase functions to destabilize kinetochore bound microtubules it may also stabilize certain slow turnover, non-kinetochore microtubules. Dissection of the dynamics of microtubule populations provides a greater understanding of mitotic spindle kinetics and insight into their roles in facilitating chromosome attachment, movement, and segregation during mitosis.

Regulation of invasion-associated actin dynamics by the Chlamydia trachomatis effectors TarP and TmeA

The obligate intracellular pathogen Chlamydia trachomatis manipulates the host actin cytoskeleton to assemble actin-rich structures that drive pathogen entry. This actin remodeling event exhibits relatively rapid dynamics that, through quantitative live-cell imaging, was revealed to consist of three phases – a fast recruitment phase which abruptly transitions to a fast turnover phase before resolving into a slow turnover of actin, indicating the end of actin remodeling. Here, we investigate Chlamydia invasion in the context of actin dynamics. Efficient invasion was associated with robust actin remodeling kinetics, which was linked to signaling from the type-III secreted effectors TarP and TmeA, and the actin nucleating activities of formin 1 (Fmn1) and Arp2/3. Stable recruitment of Fmn1 and Arp2/3 was dependent upon TarP and/or TmeA, although TarP signaling was responsible for the majority of Fmn1 and Arp2/3 recruitment. Rapid actin kinetics were due in part to a collaborative functional interaction between two different classes of actin nucleators – formins, including formin 1 and the diaphanous-related formins mDia1 and mDia2, and the Arp2/3 complex. Inhibition of either formin or Arp2/3, or deletion of TarP and TmeA, prevented this collaboration and resulted in attenuated actin kinetics and invasion efficiency. Collectively, these data support a model wherein TarP and TmeA signaling are core components of actin remodeling that operate via stable recruitment of formin and Arp2/3. At the population level, the kinetics of recruitment and turnover of actin and its nucleators were linked. However, reanalysis of the data at the level of individual elementary bodies showed significant variation and a lack of correlation between the kinetics of recruitment and turnover, suggesting that accessory factors variably modify actin kinetics at individual entry sites. In summary, efficient chlamydial invasion is an effector-driven process that requires a specific profile of actin recruitment which arises following collaboration between formin and Arp2/3.

NMDA Receptor Hypofunction in the Aging-Associated Malfunction of Peripheral Tissue

Glutamatergic transmission through NMDA receptors (NMDARs) is important for the function of peripheral tissues. In the bone, NMDARs and its co-agonist, D-serine participate in all the phases of the remodeling. In the vasculature, NMDARs exerts a tonic vasodilation decreasing blood perfusion in the corpus cavernosum and the filtration rate in the renal glomerulus. NMDARs are relevant for the skin turnover regulating the proliferation and differentiation of keratinocytes and the formation of the cornified envelope (CE). The interference with NMDAR function in the skin leads to a slow turnover and repair. As occurs with the brain and cognitive functions, the manifestations of a hypofunction of NMDARs resembles those observed during aging. This raises the question if the deterioration of the glomerular vasculature, the bone remodeling and the skin turnover associated with age could be related with a hypofunction of NMDARs. Furthermore, the interference of D-serine and the effects of its supplementation on these tissues, suggest that a decrease of D-serine could account for this hypofunction pointing out D-serine as a potential therapeutic target to reduce or even prevent the detriment of the peripheral tissue associated with aging.

The Chlamydia trachomatis type III effector TarP coordinates a functional collaboration between the actin nucleators Formin 1 and Arp2/3 during invasion

The obligate intracellular pathogen Chlamydia trachomatis manipulates the host actin cytoskeleton to assemble actin-rich structures that drive pathogen entry. This actin remodeling event exhibits relatively rapid dynamics that, through quantitative live-cell imaging, was revealed to consist of three phases – a fast recruitment phase which abruptly transitions to a fast turnover phase before resolving into a slow turnover of actin that indicates the end of actin remodeling. Here, we investigate Chlamydia invasion in the context of actin dynamics. Efficient invasion is associated with robust actin remodeling kinetics that results from a collaborative functional interaction between two different classes of actin nucleators – formins, including formin 1 and the diaphanous-related formins mDia1 and mDia2, and the Arp2/3 complex. Recruitment of these nucleators requires the presence of the chlamydial type III effector TarP, which enables the respective nucleating activities of formin and Arp2/3 to collaboratively generate a robust actin network. A collaborative model is supported by the observation that co-inhibition of Fmm1 and Arp2/3 further reduced both actin dynamics and invasion efficiency than either treatment alone. Furthermore, inhibition of recruitment of Fmn1 and/or Arp2/3 by deleting TarP was sufficient to similarly attenuated actin kinetics and invasion efficiency, supporting a model wherein TarP is the major contributor to robust actin remodeling via its recruitment of the two classes of actin nucleators. At the population level, the kinetics of recruitment and turnover of actin and its nucleators were linked. However, a more detailed analysis of the data at the level of individual elementary bodies showed significant variation and a lack of correlation between the kinetics of recruitment and turnover, suggesting that accessory factors variably modify actin kinetics at individual entry sites. In summary, efficient chlamydial invasion requires a specific profile of actin dynamics which are coordinated by TarP-dependent recruitment of two classes of actin nucleators.

Different SP1 binding dynamics at individual genomic loci in human cells

Using a tamoxifen-inducible time-course ChIP-seq approach, we show that the ubiquitous transcription factor SP1 has different binding dynamics at its target sites in the human genome that are not correlated with SP1 occupancy levels at those sites. While ~70% of SP1 binding sites are located in promoter regions, loci with slow SP1 binding turnover are enriched in enhancer and Polycomb-repressed regions. Unexpectedly, SP1 sites with fast turnover times tend to have higher quality and more copies of the SP1 sequence motif. Different co-binding factors associate near SP1 binding sites depending on their binding kinetics and on their location at promoters or enhancers. For example, NFY and FOS are preferentially associated near promoter-bound SP1 sites with fast turnover, whereas DNA motifs of ETS and homeodomain proteins are preferentially observed at sites with slow turnover. At promoters but not enhancers, proteins involved in sumoylation and PML bodies associate more strongly with slow SP1 binding sites than with the fast-binding sites. The speed of SP1 binding turnover is not associated with nucleosome occupancy, and it is not necessarily coupled to higher transcriptional activity. These results with SP1 are in contrast from those of human TBP, indicating that there is no common mechanism affecting transcription factor binding kinetics.

Nitrogen Cycling and Mass Balance in the World’s Mangrove Forests

Nitrogen (N) cycling in mangroves is complex, with rapid turnover of low dissolved N concentrations, but slow turnover of particulate N. Most N is stored in soils. The largest sources of N are nearly equal amounts of mangrove and benthic microalgal primary production. Dissolved N fluxes between the forests and tidal waters show net uptake, indicating N conservation. N2-fixation is underestimated as rapid rates measured on tree stems, aboveground roots and cyanobacterial mats cannot currently be accounted for at the whole-forest scale due to their extreme patchiness and the inability to extrapolate beyond a localized area. Net immobilization of NH4+ is the largest ecosystem flux, indicating N retention. Denitrification is the largest loss of N, equating to 35% of total N input. Burial equates to about 29% of total inputs and is the second largest loss of N. Total inputs slightly exceed total outputs, currently suggesting net N balance in mangroves. Mangrove PON export equates to ≈95% of PON export from the world’s tropical rivers, but only 1.5% of the entire world’s river discharge. Mangrove N2O emissions, denitrification, and burial contribute 0.4%, 0.5–2.0% and 6%, respectively, to the global coastal ocean, which are disproportionate to their small worldwide area.

A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming

MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target mRNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter highly complementary targets, which can trigger a process termed target-directed microRNA degradation (TDMD). During TDMD, miRNAs undergo tailing and trimming, suggesting that this is an important step in the decay mechanism. Here, we identified a cullin-RING ubiquitin ligase (CRL), containing the substrate adapter ZSWIM8, that mediated TDMD. The ZSWIM8 CRL interacted with AGO proteins, promoted TDMD in a tailing and trimming-independent manner, and regulated miRNA expression in multiple cell types. These findings suggest a model in which the ZSWIM8 ubiquitin ligase mediates TDMD by directing proteasomal decay of miRNA-containing complexes engaged with highly complementary targets.

Master curve of boosted diffusion for 10 catalytic enzymes

Molecular agitation more rapid than thermal Brownian motion is reported for cellular environments, motor proteins, synthetic molecular motors, enzymes, and common chemical reactions, yet that chemical activity coupled to molecular motion contrasts with generations of accumulated knowledge about diffusion at equilibrium. To test the limits of this idea, a critical testbed is the mobility of catalytically active enzymes. Sentiment is divided about the reality of enhanced enzyme diffusion, with evidence for and against. Here a master curve shows that the enzyme diffusion coefficient increases in proportion to the energy release rate—the product of Michaelis-Menten reaction rate and Gibbs free energy change (ΔG)—with a highly satisfactory correlation coefficient of 0.97. For 10 catalytic enzymes (urease, acetylcholinesterase, seven enzymes from the glucose cascade cycle, and one other), our measurements span from a roughly 40% enhanced diffusion coefficient at a high turnover rate and negativeΔGto no enhancement at a slow turnover rate and positiveΔG. Moreover, two independent measures of mobility show consistency, provided that one avoids undesirable fluorescence photophysics. The master curve presented here quantifies the limits of both ideas, that enzymes display enhanced diffusion and that they do not within instrumental resolution, and has possible implications for understanding enzyme mobility in cellular environments. The striking linear dependence of ΔGfor the exergonic enzymes (ΔG<0), together with the vanishing effect for endergonic enzyme (ΔG>0), are consistent with a physical picture in which the mechanism boosting the diffusion is an active one, utilizing the available work from the chemical reaction.

Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway

Nuclear pore complexes (NPCs) are important for cellular functions beyond nucleocytoplasmic trafficking, including genome organization and gene expression. This multi-faceted nature and the slow turnover of NPC components complicates investigations of how individual nucleoporins act in these diverse processes. To address this question, we apply an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins NUP153, NUP50 and TPR. Acute depletion of TPR causes rapid and pronounced changes in transcriptomic profiles. These changes are dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover.

Displacement of Slow-Turnover DNA Glycosylases by Molecular Traffic on DNA

In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1–DNA product complex was disrupted by DNA polymerase β (POLβ) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLβ and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.