Were eukaryotes made by sex?: Sex might have been vital for merging endosymbiont and host genomes giving rise to eukaryotes.

I hypothesize that the appearance of sex facilitated the merging of the endosymbiont and host genomes during early eukaryote evolution. Eukaryotes were formed by symbiosis between a bacterium that entered an archaeon, eventually giving rise to mitochondria. This entry was followed by the gradual transfer of most bacterial endosymbiont genes into the archaeal host genome. I argue that the merging of the mitochondrial genes into the host genome was vital for the evolution of genuine eukaryotes. At the time this process commenced it was unprecedented and required a novel mechanism. I suggest that this mechanism was meiotic sex, and that its appearance might have been THE crucial step that enabled the evolution of proper eukaryotes from early endosymbiont containing proto-eukaryotes. Sex might continue to be essential today for keeping genome insertions in check. Also see the video abstract here: https://youtu.be/aVMvWMpomac.

New-age ideas about age-old sex: separating meiosis from mating could solve a century-old conundrum.

Ever since Darwin first addressed it, sexual reproduction reigns as the 'queen' of evolutionary questions. Multiple theories tried to explain how this apparently costly and cumbersome method has become the universal mode of eukaryote reproduction. Most theories stress the adaptive advantages of sex by generating variation, they fail however to explain the ubiquitous persistence of sexual reproduction also where adaptation is not an issue. I argue that the obstacle for comprehending the role of sex stems from the conceptual entanglement of two distinct processes - gamete production by meiosis and gamete fusion by mating (mixis). Meiosis is an ancient, highly rigid and evolutionary conserved process identical and ubiquitous in all eukaryotes. Mating, by contrast, shows tremendous evolutionary variability even in closely related clades and exhibits wonderful ecological adaptability. To appreciate the respective roles of these two processes, which are normally linked and alternating, we require cases where one takes place without the other. Such cases are rather common. The heteromorphic sex chromosomes Y and W, that do not undergo meiotic recombination are an evolutionary test case for demonstrating the role of meiosis. Substantial recent genomic evidence highlights the accelerated rates of change and attrition these chromosomes undergo in comparison to those of recombining autosomes. I thus propose that the most basic role of meiosis is conserving integrity of the genome. A reciprocal case of meiosis without bi-parental mating, is presented by self-fertilization, which is fairly common in flowering plants, as well as most types of apomixis. I argue that deconstructing sex into these two distinct processes - meiosis and mating - will greatly facilitate their analysis and promote our understanding of sexual reproduction.

Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response.

Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

Slip slidin' away of mitosis with CRL2Zyg11.

The spindle assembly checkpoint arrests mitotic cells by preventing degradation of cyclin B1 by the anaphase-promoting complex/cyclosome, but some cells evade this checkpoint and slip out of mitosis. Balachandran et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601083) show that the E3 ligase CRL2ZYG11 degrades cyclin B1, allowing mitotic slippage.

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.

Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.

Imaging Cell Cycle Phases and Transitions of Living Cells from Yeast to Woman.

The eukaryotic cell cycle is comprised of different phases that take place sequentially once, and normally only once, every division cycle. Such a dynamic process is best viewed in real time in living dividing cells. The insights that can be gained from such methods are considerably larger than any alternative technique that only generates snapshots. A great number of studies can gain from live cell imaging; however this method often feels somewhat intimidating to the novice. The purpose of this chapter is to demonstrate that imaging cell cycle phases in living cells from yeast to human is relatively easy and can be performed with equipment that is available in most research institutes. We present the different approaches, review different types of reporters, and discuss in depth all the aspects to be considered to obtain optimal results. We also describe our latest cell cycle markers, which afford unprecedented "sub"-phase temporal resolution.

Phosphorylation and dephosphorylation regulate APC/C(Cdh1) substrate degradation.

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C(Cdh1) mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1(m11) mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C(Cdh1) substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.

Degradation of Ndd1 by APC/C(Cdh1) generates a feed forward loop that times mitotic protein accumulation.

Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C(Cdh1) substrates. The APC/C(Cdh1) thus regulates these proteins in a dual manner­both pretranscriptionally and post-translationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C(Cdh1) inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C(Cdh1) substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C(Cdh1) activity is converted into multiple outputs by interactions between its substrates.

Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations.

Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP-Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases.

G0-G1 transition and the restriction point in pancreatic ß-cells in vivo.

Most of our knowledge on cell kinetics stems from in vitro studies of continuously dividing cells. In this study, we determine in vivo cell-cycle parameters of pancreatic ß-cells, a largely quiescent population, using drugs that mimic or prevent glucose-induced replication of ß-cells in mice. Quiescent ß-cells exposed to a mitogenic glucose stimulation require 8 h to enter the G1 phase of the cell cycle, and this time is prolonged in older age. The duration of G1, S, and G2/M is ~5, 8, and 6 h, respectively. We further provide the first in vivo demonstration of the restriction point at the G0-G1 transition, discovered by Arthur Pardee 40 years ago. The findings may have pharmacodynamic implications in the design of regenerative therapies aimed at increasing ß-cell replication and mass in patients with diabetes.

Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting.

Ubiquitin accumulation in amyloid plaques is a pathological marker observed in the vast majority of neurodegenerative diseases, yet ubiquitin function in these inclusions is controversial. It has been suggested that ubiquitylated proteins are directed to inclusion bodies under stress conditions, when both chaperone-mediated refolding and proteasomal degradation are compromised or overwhelmed. Alternatively, ubiquitin and chaperones may be recruited to preformed inclusions to promote their elimination. We address this issue using a yeast model system, based on expression of several mildly misfolded degradation substrates in cells with altered chaperone content. We find that the heat shock protein 70 (Hsp70) chaperone pair Ssa1/Ssa2 and the Hsp40 cochaperone Sis1 are essential for degradation. Substrate ubiquitylation is strictly dependent on Sis1, whereas Ssa1 and Ssa2 are dispensable. Remarkably, in Ssa1/Ssa2-depleted cells, ubiquitylated substrates are sequestered into detergent-insoluble, Hsp42-positive inclusion bodies. Unexpectedly, sequestration is abolished by preventing substrate ubiquitylation. We conclude that Hsp40 is required for the targeting of misfolded proteins to the ubiquitylation machinery, whereas the decision to degrade or sequester ubiquitylated proteins is mediated by the Hsp70s. Accordingly, diminished Hsp70 levels, as observed in aging or certain pathological conditions, might be sufficient to trigger ubiquitin-dependent sequestration of partially misfolded proteins into inclusion bodies.

A transgenic mouse marking live replicating cells reveals in vivo transcriptional program of proliferation.

Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of replicating cells in their in vivo niche.

Indirect inhibition of 26S proteasome activity in a cellular model of Huntington's disease.

Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington's disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt-whether aggregated or not-did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network.

Phosphorylation of Cdc5 regulates its accumulation.

BACKGROUND: Cdc5 (polo kinase/Plk1) is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28) phosphorylation site present. FINDINGS: Cdc5T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans) and drastically reduces Clb2 levels. CONCLUSIONS: Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1) caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.

Leishmania express a functional Cdc20 homologue.

Our knowledge concerning the mechanisms of cell cycle regulation in organisms belonging to the Trypanosometidae family is limited. Leishmania donovani are parasitic protozoa that cause kala azar, a fatal form of visceral leishmaniasis in humans. Here we provide evidence that the L. donovani genome contains a Cdc20 homologue. Cdc20 is a regulator of the Anaphase Promoting Complex/Cyclosome (APC/C) that mediates ubiquitin-dependent proteasomal degradation of key cell cycle regulators in eukaryotes. We show that L. donovani Cdc20 protein (LdCdc20p) can complement a lack of yeast Cdc20 protein in Saccharomyces cerevisiae cells, validating the functionality of LdCdc20p. Furthermore, we demonstrate cyclic expression of LdCdc20p and that it contains an active RXXL destruction motif, a distinctive feature of proteins targeted for proteasomal degradation by APC/C. Finally, in line with the proteasome mediating LdCdc20p degradation, promastigotes exposed to proteasome inhibitor display elevated LdCdc20p levels. Taken together our data indicate that Leishmania regulate their cell cycle by ubiquitin-dependent proteasomal degradation mediated by the APC/C.

The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr.

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil(-/-) embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint.

Cdk1 and SUMO regulate Swe1 stability.

The Swe1/Wee1 kinase phosphorylates and inhibits Cdk1-Clb2 and is a major mitotic switch. Swe1 levels are controlled by ubiquitin mediated degradation, which is regulated by interactions with various mitotic kinases. We have recently reported that Swe1 levels are capable of sensing the progress of the cell cycle by measuring the levels of Cdk1-Clb2, Cdc5 and Hsl1. We report here a novel mechanism that regulates the levels of Swe1. We show that S. cerevisiae Swe1 is modified by Smt3/SUMO on residue K594 in a Cdk1 dependant manner. A degradation of the swe1(K594R) mutant that cannot be modified by Smt3 is considerably delayed in comparison to wild type Swe1. Swe1(K594R) cells express elevated levels of Swe1 protein and demonstrate higher levels of Swe1 activity as manifested by Cdk1-Y19 phosphorylation. Interestingly this mutant is not targeted, like wild type Swe1, to the bud neck where Swe1 degradation takes place. We show that Swe1 is SUMOylated by the Siz1 SUMO ligase, and consequently siz1Δ cells express elevated levels of Swe1 protein and activity. Finally we show that swe1(K594R) cells are sensitive to osmotic stress, which is in line with their compromised regulation of Swe1 degradation.

Clb2 and the APC/C(Cdh1) regulate Swe1 stability.

Swe1/Wee1 regulates mitotic entry by inhibiting Clb2-Cdk1 and its accumulation is involved in stress induced G(2) arrest. The APC/C(Cdh1) substrates Cdc5, Clb2 and Hsl1 regulate Swe1 degradation. We observed that clb2Deltacdh1Delta double mutant S. cerevisiae does not express any detectable levels of Swe1, presumably due to its constitutive degradation. This effect of Cdh1 inactivation is due to stabilization of Cdc5 and Hsl1, as expression of the non-degradable Cdc5(T29A) in clb2Delta cells prevented Swe1 accumulation. Strikingly, expression of non-degradable Hsl1(mdb/mkb) prevented Swe1 accumulation even in wild type Clb2 cells. Interestingly Swe1 accumulation could be reconstituted in all these mutants by eliciting a replication fork stress with hydroxyurea. Cells expressing the Clb2(ME) mutant, that cannot bind Swe1, behaved like clb2Delta cells, and failed to accumulate Swe1 in the absence of Cdh1 or the presence of Cdc5(T29A). This suggests that for Swe1 to accumulate it must interact with Clb2. We further show that in the absence of Clb2, Hsl1 is no longer essential for Swe1 degradation. We hypothesize that Clb2-Cdk1 protects Swe1 from premature degradation until its Hsl1 mediated de-protection, which enables its Cdc5 mediated degradation. Swe1 levels are thus regulated by monitoring the levels of three major mitotic regulators.

Fifteen years of APC/cyclosome: a short and impressive biography.

The APC/C (anaphase-promoting complex/cyclosome) discovered exactly 15 years ago by Avram Heshko and Marc Kirschner is by far the most complex ubiquitin ligase discovered so far. The APC/C is composed of roughly a dozen subunits and measures a massive 1.5 MDa. This huge complex, as well as its multiple modes of regulation, boasts impressive evolutionary conservation. One of its most puzzling features is its split personality: regulation of mitotic exit events on the one hand, and its ongoing activity during G(1)-phase, G(0)-phase and in terminally differentiated cells. The present short review is intended to provide a basic description of our current understanding of the APC/C, focusing on recent findings concerning its role in G(1)-phase and in differentiated cells.

APC/CCdh1 specific degradation of Hsl1 and Clb2 is required for proper stress responses of S. cerevisiae.

Cdh1 activates the Anaphase Promoting Complex/Cyclosome (APC/C(Cdh1)) throughout G(1) to degrade key cell cycle proteins. Cdh1 is not essential for cell proliferation, in spite of the fact that overexpression of some its degradation substrates is highly toxic. We report here that cdh1Delta cells are sensitive to stresses that activate the CWI (Cell Wall Integrity) and Hog1 MAP kinase pathways. Stresses did not activate APC/C(Cdh1) and cellular sensitivity was thus clearly due to constitutively elevated substrate levels. To explore the contribution of stabilization of individual APC/C(Cdh1) substrates to stress sensitivity, we generated cell lines expressing stabilized substrate mutants under their endogenous promoters. Cells expressing stabilized Hsl1 were sensitive to caffeine and failed to activate the Slt2 pathway. Cells expressing partially stable Clb2 were particularly sensitive to different stresses, possibly due to reduced Sic1 levels. Cells expressing stabilized Cdc5 were much less stress sensitive. Interestingly sensitivity of cdh1Delta cells does not seem to be restricted to G(1) but is manifested also during S and G(2) when the APC/C(Cdh1) is inactive anyway. We thus hypothesize that a role of G(1) specific APC/C(Cdh1) activity is to reset substrate levels to enables appropriate regulation of substrate accumulation in the subsequent phases of the cell cycle.