Asymmetric chromatin retention and nuclear envelopes separate chromosomes in fused cells in vivo.

Hybrid cells derived through fertilization or somatic cell fusion recognize and separate chromosomes of different origins. The underlying mechanisms are unknown but could prevent aneuploidy and tumor formation. Here, we acutely induce fusion between Drosophila neural stem cells (neuroblasts; NBs) and differentiating ganglion mother cells (GMCs) in vivo to define how epigenetically distinct chromatin is recognized and segregated. We find that NB-GMC hybrid cells align both endogenous (neuroblast-origin) and ectopic (GMC-origin) chromosomes at the metaphase plate through centrosome derived dual-spindles. Physical separation of endogenous and ectopic chromatin is achieved through asymmetric, microtubule-dependent chromatin retention in interphase and physical boundaries imposed by nuclear envelopes. The chromatin separation mechanisms described here could apply to the first zygotic division in insects, arthropods, and vertebrates or potentially inform biased chromatid segregation in stem cells.

Principles and mechanisms of asymmetric cell division.

Asymmetric cell division (ACD) is an evolutionarily conserved mechanism used by prokaryotes and eukaryotes alike to control cell fate and generate cell diversity. A detailed mechanistic understanding of ACD is therefore necessary to understand cell fate decisions in health and disease. ACD can be manifested in the biased segregation of macromolecules, the differential partitioning of cell organelles, or differences in sibling cell size or shape. These events are usually preceded by and influenced by symmetry breaking events and cell polarization. In this Review, we focus predominantly on cell intrinsic mechanisms and their contribution to cell polarization, ACD and binary cell fate decisions. We discuss examples of polarized systems and detail how polarization is established and, whenever possible, how it contributes to ACD. Established and emerging model organisms will be considered alike, illuminating both well-documented and underexplored forms of polarization and ACD.

Aggresome-Like Formation Promotes Resistance to Proteotoxicity in Cells from Long-Lived Species.

The capacity of cells to maintain proteostasis declines with age, causing rapid accumulation of damaged proteins and protein aggregates, which plays an important role in age-related disease etiology. While our group and others have identified that proteostasis is enhanced in long-lived species, there are no data on whether this leads to better resistance to proteotoxicity. We compared the sensitivity of cells from long- (naked mole rat [NMR]) and short- (Mouse) lived species to proteotoxicity, by measuring the survival of fibroblasts under polyglutamine (polyQ) toxicity, a well-established model of protein aggregation. Additionally, to evaluate the contribution of proteostatic mechanisms to proteotoxicity resistance, we down-regulated a key protein of each mechanism (autophagy-ATG5; ubiquitin-proteasome-PSMD14; and chaperones-HSP27) in NMR fibroblasts. Furthermore, we analyzed the formation and subcellular localization of inclusions in long- and short-lived species. Here, we show that fibroblasts from long-lived species are more resistant to proteotoxicity than their short-lived counterparts. Surprisingly, this does not occur because the NMR cells have less polyQ82 protein aggregates, but rather they have an enhanced capacity to handle misfolded proteins and form protective perinuclear and aggresome-like inclusions. All three proteostatic mechanisms contribute to this resistance to polyQ toxicity but autophagy has the greatest effect. Overall, our data suggest that the resistance to proteotoxicity observed in long-lived species is not due to a lower level of protein aggregates but rather to enhanced handling of the protein aggregates through the formation of aggresome-like inclusions, a well-recognized protective mechanism against proteotoxicty.

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism.

Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-ß-galactosidase (ß-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased ß-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in ß-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.

Rapamycin and the inhibition of the secretory phenotype.

Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Cell senescence is a complex process that can be triggered by multiple challenges. Recently it has been observed that the composition of the secretory phenotype or SASP depends on the insult that triggers cell senescence. Rapamycin, an inhibitor of mTOR that increases longevity in several species, inhibits cell senescence in vitro, while silencing the Nrf2 gene induces premature senescence. We have found that rapamycin activates the Nrf2 pathway to regulate cell cycle arrest, but not the production of SASP, which is regulated by a different pathway, probably involving the inhibition of MAPKAPK2.

Gene expression in the liver of female, but not male mice treated with rapamycin resembles changes observed under dietary restriction.

It is well known that in mice the extension in lifespan by rapamycin is sexually dimorphic, in that it has a larger effect in females than males. In a previous study we showed that in male C57BL6 mice, rapamycin had less profound effects in both gene expression and liver metabolites when compared to dietary restriction (DR), but no data was available in females. Because recent studies showed that rapamycin increases longevity in a dose dependent manner and at every dose tested the effect remains larger in females than in males, we hypothesized that rapamycin should have a stronger effect on gene expression in females, and this effect could be dose dependent. To test this hypothesis, we measured the changes in liver gene expression induced by rapamycin (14 ppm) with a focus on several genes involved in pathways known to play a role in aging and that are altered by DR. To investigate whether any effects are dose dependent, we also analyzed females treated with two additional doses of rapamycin (22 and 42 ppm). We observed striking differences between male and female in gene expression at 14 ppm, where females have a larger response to rapamycin than males, and the effects of rapamycin in females resemble what we observed under DR. However, these effects were generally not dose dependent. These data support the notion that female mice respond better to rapamycin, and at least with the set of genes studied here, the effect of rapamycin in females resemble the effect of DR.

Long-lived species have improved proteostasis compared to phylogenetically-related shorter-lived species.

Our previous studies have shown that the liver from Naked Mole Rats (NMRs), a long-lived rodent, has increased proteasome activity and lower levels of protein ubiquitination compared to mice. This suggests that protein quality control might play a role in assuring species longevity. To determine whether enhanced proteostasis is a common mechanism in the evolution of other long-lived species, here we evaluated the major players in protein quality control including autophagy, proteasome activity, and heat shock proteins (HSPs), using skin fibroblasts from three phylogenetically-distinct pairs of short- and long-lived mammals: rodents, marsupials, and bats. Our results indicate that in all cases, macroautophagy was significantly enhanced in the longer-lived species, both at basal level and after induction by serum starvation. Similarly, basal levels of most HSPs were elevated in all the longer-lived species. Proteasome activity was found to be increased in the long-lived rodent and marsupial but not in bats. These observations suggest that long-lived species may have superior mechanisms to ensure protein quality, and support the idea that protein homeostasis might play an important role in promoting longevity.

Conservation of the three-dimensional structure in non-homologous or unrelated proteins.

In this review, we examine examples of conservation of protein structural motifs in unrelated or non-homologous proteins. For this, we have selected three DNA-binding motifs: the histone fold, the helix-turn-helix motif, and the zinc finger, as well as the globin-like fold. We show that indeed similar structures exist in unrelated proteins, strengthening the concept that three-dimensional conservation might be more important than the primary amino acid sequence.

Identification of a small molecule PriA helicase inhibitor.

PriA helicase catalyzes the initial steps of replisome reloading onto repaired DNA replication forks in bacterial DNA replication restart pathways. We have used a high-throughput screen to identify a small molecule inhibitor of PriA-catalyzed duplex DNA unwinding. The compound, CGS 15943, targets Neisseria gonorrhoeae PriA helicase with an IC(50) of 114 ± 24 µM. The PriA helicase of Escherichia coli is also inhibited, although to a lesser extent than N. gonorrhoeae PriA. CGS 15943 decreases rates of PriA-catalyzed ATP hydrolysis and reduces the affinity with which PriA binds DNA. Steady-state kinetic data indicate that CGS 15943 inhibits PriA through a mixed mode of inhibition with respect to ATP and with respect to DNA, indicating that it binds to a site on PriA that participates in both substrate binding and catalysis. Inhibitor binding constants derived from steady-state kinetic experiments reveal that CGS 15943 has the highest binding affinity for the PriA·PriB·ATP complex, intermediate binding affinity for the PriA·PriB·DNA complex, and the lowest binding affinity for the PriA·PriB·DNA·ATP complex, suggesting that PriA assumes different conformations in each of these complexes. We propose that CGS 15943 binds to PriA at a site distinct from the DNA and primary ATP binding sites, perhaps at PriA's weak nucleotide binding site, and induces a conformational change in PriA that renders it less catalytically proficient or prevents conformational changes in PriA that are necessary for ATP hydrolysis and duplex DNA unwinding.

A bacterial PriB with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase.

BACKGROUND: Bacterial DNA replication restart pathways facilitate reinitiation of DNA replication following disruptive encounters of a replisome with DNA damage, thereby allowing complete and faithful duplication of the genome. In Neisseria gonorrhoeae, the primosome proteins that catalyze DNA replication restart differ from the well-studied primosome proteins of E. coli with respect to the number of proteins involved and the affinities of their physical interactions: the PriA:PriB interaction is weak in E. coli, but strong in N. gonorrhoeae, and the PriB:DNA interaction is strong in E. coli, but weak in N. gonorrhoeae. In this study, we investigated the functional consequences of this affinity reversal. RESULTS: We report that N. gonorrhoeae PriA's DNA binding and unwinding activities are similar to those of E. coli PriA, and N. gonorrhoeae PriA's helicase activity is stimulated by its cognate PriB, as it is in E. coli. This finding is significant because N. gonorrhoeae PriB's single-stranded DNA binding activity is weak relative to that of E. coli PriB, and in E. coli, PriB's single-stranded DNA binding activity is important for PriB stimulation of PriA helicase. Furthermore, a N. gonorrhoeae PriB variant defective for binding single-stranded DNA can stimulate PriA's helicase activity, suggesting that DNA binding by PriB might not be important for PriB stimulation of PriA helicase in N. gonorrhoeae. We also demonstrate that N. gonorrhoeae PriB stimulates ATP hydrolysis catalyzed by its cognate PriA. This activity of PriB has not been observed in E. coli, and could be important for PriB stimulation of PriA helicase in N. gonorrhoeae. CONCLUSIONS: The results of this study demonstrate that a bacterial PriB homolog with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase. While it remains unclear if N. gonorrhoeae PriB's weak DNA binding activity is required for PriB stimulation of PriA helicase, the ability of PriB to stimulate PriA-catalyzed ATP hydrolysis could play an important role. Thus, the weak interaction between N. gonorrhoeae PriB and DNA might be compensated for by the strong interaction between PriB and PriA, which could result in allosteric activation of PriA's ATPase activity.