Chromosome segregation during female meiosis in C. elegans: A tale of pushing and pulling

The role of the kinetochore during meiotic chromosome segregation in C. elegans oocytes has been a matter of controversy. Danlasky et al. (2020. J. Cell. Biol. https://doi.org/10.1083/jcb.202005179) show that kinetochore proteins KNL-1 and KNL-3 are required for early stages of anaphase during female meiosis, suggesting a new kinetochore-based model of chromosome segregation.

Meiosis consists of two consecutive chromosome segregation events preceded by a single round of DNA replication. Homologous chromosomes are separated in meiosis I, which is followed by sister chromatid separation in meiosis II to produce haploid gametes. Both of these stages require chromosomes/chromatids to align during metaphase before separating to opposite poles during anaphase. During mitosis, microtubules emanating from centrosomes at opposite poles of the cell bind chromosomes through a multiprotein complex called the kinetochore, allowing chromosomes to be pulled apart (1, 2). This segregation event takes place in two stages: anaphase A, where chromosomes are pulled toward spindle poles due to microtubule depolymerization, and anaphase B, where spindle poles themselves move farther apart, taking the attached chromosomes with them (3, 4). In many organisms, including mammals, oocytes lack centrosomes, and it has been of great interest to clarify the mechanisms used to ensure chromosomes are properly segregated during female meiosis (5, 6). Caenorhabditis elegans has served as a model for studying both mitosis and meiosis, but the mechanisms operating during female meiosis have been a matter of debate and controversy.In 2010, Dumont et al. showed that the kinetochore is required for chromosome alignment and congression during metaphase (7). However, they suggested that chromosome segregation was the result of microtubule polymerization between the segregating chromosomes (Fig. 1), resulting in a pushing force exerted onto chromosomes toward the spindle poles in a largely kinetochore-independent manner (7). This mechanism was also supported by the finding that CLIP-associated protein (CLASP)–dependent microtubule polymerization between the segregating chromosomes is essential for chromosome separation (8). An alternative model suggested that chromosomes are transported through microtubule-free channels toward the spindle poles by the action of dynein (9). Later evidence put in doubt a role for dynein and favored a model in which chromosomes initially separate when the spindle shortens and the poles overlap with chromosomes in an anaphase A–like mechanism. This is then followed by separation of chromosome-bound poles by outward microtubule sliding in an anaphase B–like fashion (10). However, because microtubules emanating from the spindle poles are not required to separate the homologous chromosomes but microtubules between the separating chromosomes are (8), this model is unlikely, at least as an explanation for mid-/late-anaphase movement. Furthermore, although lateral microtubule interactions with chromosomes predominate during metaphase of C. elegans oocyte meiosis, cryo-electron tomography data described end-on attachments between the separating chromosomes as anaphase progresses (11). This led to the suggestion that lateral microtubule interactions with chromosomes are responsible for the initial separation, but microtubule polymerization between the separating chromosomes is required for the later stages of segregation (11). The mechanisms involved in this initial separation have remained obscure. In this issue, Danlasky et al. show that the kinetochore is in fact required for the initial stages of chromosome segregation during female meiosis—an important step forward in our understanding of the mechanisms governing acentrosomal chromosome segregation (12).Open in a separate windowFigure 1.Some of the key findings in Danlasky et al. Kinetochore proteins surround the outer surface of the chromosomes, resulting in a characteristic cup shape. As anaphase progresses, chromosomes come into close contact to the spindle poles (anaphase A). Chromosome stretching is provided by KNL-1, MIS-12 (KNL-3), and NDC-80 (KMN)–dependent forces. Once the spindle starts elongating (anaphase B), central spindle microtubules provide the pushing forces for chromosome segregation. At this stage, kinetochore proteins also occupy the inward face of separating chromosomes. Upon KMN network depletion, bivalents flatten, and chromosome congression and alignment are defective. Anaphase A chromosome movement is almost absent, which leads to error-prone anaphase B.By simultaneously depleting kinetochore proteins KNL-1 and KNL-3 in C. elegans, Danlasky et al. observed the meiotic chromosome congression and alignment defects described in previous studies (7). However, this double-depletion phenotype displayed three key characteristics that suggested a role for kinetochores in chromosome segregation, which are discussed below.The kinetochore is required for bivalent stretching. It was previously shown that the bivalent chromosomes stretch before the initiation of segregation (10). Danlasky et. al found that this stretching of the chromosomes did not occur when KNL-1,3 were depleted, indicating that the kinetochore is required for this process (Fig. 1). Together with the observation that kinetochore proteins appear to extend toward the spindle poles, this finding suggested that pulling forces resulting from the interaction between the kinetochore and spindle microtubules are occurring during metaphase/preanaphase (Fig. 1).The kinetochore is required for anaphase A. In C. elegans female meiosis, anaphase A occurs when homologous chromosomes begin to separate during spindle shortening, and anaphase B when the chromosomes separate alongside the spindle poles (10). Danlasky et al. observed that KNL-1,3 depletion drastically reduced the velocity of anaphase A, as chromosomes only separated when spindle poles began to move apart. This indicated that pulling forces caused by the interaction between the kinetochore and spindle microtubules are also important for the initial separation of homologous chromosomes in anaphase A.The kinetochore is required for proper separation of homologous chromosomes. In KNL-1,3 depletion strains, 60% of bivalents failed to separate before segregation began, resulting in intact bivalents being pulled to the same spindle pole (Fig. 1). This failure of homologous chromosomes to separate was not thought to be a result of KNL-1,3 depletion interfering with the cleavage of cohesin that holds the two homologous chromosomes together because (a) separase and AIR-2^AuroraB, both of which are required for cohesin cleavage, localized normally during metaphase and anaphase, and (b) bivalents separated by metaphase II. This leaves the possibility open that the failure of bivalents to separate was due to the disrupted pulling forces thought to be important in bivalent stretching and anaphase A.Altogether, these data strongly indicate that the kinetochore is required not only for chromosome congression and alignment but also for the early stages of homologue separation. Anaphase B occurred successfully in the absence of KNL-1,3 but was more error prone, likely as a result of the earlier congression and anaphase A defects. While it is clear that chromosome masses do segregate in the absence of the kinetochore, this segregation is highly erroneous as a result of defects during the earlier stages of segregation in anaphase A (Fig. 1).The findings of Danlasky et al. raise testable hypotheses that could significantly enhance our understanding of acentrosomal chromosome segregation. Further investigation of the proposed pulling forces required during metaphase and early anaphase will be of great interest. Additionally, a more detailed analysis of the dynamic localization of separase and Securin, as well as assessing successful cohesin cleavage when KNL-1,3 are depleted, would back up the assertion that the failure of homologous chromosomes to separate was not due to the kinetochore impacting cohesin cleavage. It has previously been shown that the CLASP orthologue CLS-2 in C. elegans localizes to the kinetochore surrounding the bivalent chromosomes during metaphase before relocalizing to the central spindle during anaphase (7, 8, 13). It will be interesting to examine whether this key microtubule-stabilizing protein contributes to anaphase A pulling forces alongside its essential role in microtubule polymerization between chromosomes in anaphase B (8).While the regulation of proper chromosome segregation during acentrosomal meiosis in C. elegans is not yet fully understood, Danlasky et al.’s results represent a significant step forward in this endeavor by showing that the kinetochore is in fact required for the early stages of chromosome segregation.     

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD⁺ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.     

Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.     

Environmentally induced phenotypic plasticity and DNA methylation changes in a wild potato growing in two contrasting Andean experimental gardens

DNA methylation can be environmentally modulated and plays a role in phenotypic plasticity. To understand the role of environmentally induced epigenetic variation and its dynamics in natural populations and ecosystems, it is relevant to place studies in a real-world context. Our experimental model is the wild potato Solanum kurtzianum, a close relative of the cultivated potato S. tuberosum. It was evaluated in its natural habitat, an arid Andean region in Argentina characterised by spatial and temporal environmental fluctuations. The dynamics of phenotypic and epigenetic variability (with Methyl Sensitive Amplified Polymorphism markers, MSAP) were assayed in three genotypes across three growing seasons. These genotypes were cultivated permanently and also reciprocally transplanted between experimental gardens (EG) differing in ca. 1000 m of altitude. In two seasons, the genotypes presented differential methylation patterns associated to the EG. In the reciprocal transplants, a rapid epigenomic remodelling occurred according to the growing season. Phenotypic plasticity, both spatial (between EGs within season) and temporal (between seasons), was detected. The epigenetic and phenotypic variability was positively correlated. The lack of an evident mitotic epigenetic memory would be a common response to short-term environmental fluctuations. Thus, the environmentally induced phenotypic and epigenetic variation could contribute to populations persistence through time. These results have implications for understanding the great ecological diversity of wild potatoes.     

Cross-scale quality assessment of a mechanistic cation exchange chromatography model

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing-scale can be affected by variability in feed composition, mobile phase and resin properties, or dispersion effects depending on the chromatography system at hand. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. In this study, a multicomponent steric mass-action (SMA) adsorption model was applied to the scale-up of a CEX polishing step. Based on chromatograms and elution pool data ranging from laboratory- to manufacturing-scale, the proposed modeling workflow enabled early identification of differences between scales, for example, system dispersion effects or ionic capacity variability. A multistage model qualification approach was introduced to measure the model quality and to understand the model's limitations across scales. The experimental SDM and the in silico model were qualified against large-scale data using the identical state of the art equivalence testing procedure. The mechanistic chromatography model avoided limitations of the SDM by capturing effects of bed height, loading density, feed composition, and mobile phase properties. The results demonstrate the applicability of mechanistic chromatography models as a possible alternative to conventional SDM approaches.     

A depth-dependent model of the pericellular microenvironment of chondrocytes in articular cartilage

Experimental studies suggest that the magnitude of chondrocyte deformation is much smaller than expected based on the material properties of extracellular matrix (ECM) and cells, and that this result could be explained by a structural unit, the chondron, that is thought to protect chondrocytes from large deformations in situ. We extended an existing numerical model of chondrocyte, ECM and pericellular matrix (PCM) to include depth-dependent structural information. Our results suggest that superficial zone chondrocytes, which lack a pericellular capsule (PC), are relatively stiff, and therefore are protected from excessive deformations, whereas middle and deep zone chondrocytes are softer but are protected by the PC that limits cell deformations in these regions. We conclude that cell deformations sensitively depend on the immediate structural environment of the PCM in a depth-dependent manner, and that the functional stiffness of chondrocytes in situ is much larger than experiments on isolated cells would suggest.     

Scaling-up of a B-phycoerythrin production and purification bioprocess involving aqueous two-phase systems: Practical experiences

One of the most attractive segments in food and cosmetic industries is that of natural pigments. Since some synthetic pigments have been reported to be hazardous for humans, natural pigments obtained through biotechnological processes represent an attractive alternative. Our research group has previously worked on the development of an aqueous two-phase system (ATPS)-based prototype process for the recovery of B-phycoerythrin (BPE), a natural high-value pigment obtained from Porphyridium cruentum. Detailed studies describing the scaling up of ATPS processes from bench scale to pilot plant facilities are not common. In this paper experiences derived from the scale-up of a previously developed process for production and recovery of highly purified (purity defined as the absorbance ratio A₅₄₅/A₂₈₀ > 4) BPE are described, where a scale-up factor of 850× was implemented. Characterization of cell disruption with a pilot-scale bead mill allowed efficient BPE release at 2900 rpm, 10% (w/v) sample load, 60% (v/v) bead load and 0.5 mm glass beads and 22 min of residence time with a yield of 1.35 mg BPE/g of wet biomass. BPE was recovered and purified using a strategy comprising isoelectric precipitation, aqueous two-phase fractionation and ultrafiltration. A 54% global BPE recovery yield, with final purity of 4.1, was achieved under optimal process conditions. Considering total costs for raw materials and energy expenditures for one batch, it was determined that the production cost of BPE was of $1.17 USD/mg, which is underneath the commercial price of a BPE standard (>$30 USD/mg).     

Environmental gradients shift the direction of the relationship between native and alien plant species richness

Aim

To assess how environmental, biotic and anthropogenic factors shape native–alien plant species richness relationships across a heterogeneous landscape.

Location

Banks Peninsula, New Zealand.

Methods

We integrated a comprehensive floristic survey of over 1200 systematically located 6 × 6 m plots, with corresponding climate, environmental and anthropogenic data. General linear models examined variation in native and alien plant species richness across the entire landscape, between native‐ and alien‐dominated plots, and within separate elevational bands.

Results

Across all plots, there was a significant negative correlation between native and alien species richness, but this relationship differed within subsets of the data: the correlation was positive in alien‐dominated plots but negative in native‐dominated plots. Within separate elevational bands, native and alien species richness were positively correlated at lower elevations, but negatively correlated at higher elevations. Alien species richness tended to be high across the elevation gradient but peaked in warmer, mid‐ to low‐elevation sites, while native species richness increased linearly with elevation. The negative relationship between native and alien species richness in native‐dominated communities reflected a land‐use gradient with low native and high alien richness in more heavily modified native‐dominated vegetation. In contrast, native and alien richness were positively correlated in very heavily modified alien‐dominated plots, most likely due to covariation along a gradient of management intensity.

Main conclusions

Both positive and negative native–alien richness relationships can occur across the same landscape, depending on the plant community and the underlying human and environmental gradients examined. Human habitat modification, which is often confounded with environmental variation, can result in high alien and low native species richness in areas still dominated by native species. In the most heavily human modified areas, dominated by alien species, both native and alien species may be responding to similar underlying gradients.

     

Biology and ecology of Brazilian elodea (Egeria densa) and its specific herbivore, Hydrellia sp., in Argentina

Egeria densa (Hydrocharitaceae) is a submerged macrophyte from South America that is a weed in several countries. It crowds out native plants and hinders water use, causing economic and environmental damage. The leafminer fly Hydrellia sp. 1 (Diptera: Ephydridae), was found feeding in E. densa throughout its Argentine distribution, and is currently the only known specialist herbivore of E. densa. It was reared in the laboratory and tested on 25 plant species. This herbivore can cause heavy defoliation in the laboratory and in the field. Hydrellia sp. 1 was found only on E. densa, but in the laboratory it also developed on two other Hydrocharitaceae species in the same family; Egeria naias, and Elodea callitrichoides. Significant oviposition and feeding were only observed on its primary natural host, and to a lesser degree on E. naias. Field studies indicate Hydrellia sp. 1 is present in the field year round, unless the host plant is prostrate for long periods, or covered by floating macrophytes. These results indicate Hydrellia sp. 1 may be a suitable biocontrol candidate for E. densa.