Does the Pachytene Checkpoint, a Feature of Meiosis, Filter Out Mistakes in Double-Strand DNA Break Repair and as a side-Effect Strongly Promote Adaptive Speciation?

This essay aims to explain two biological puzzles: why eukaryotic transcription units are composed of short segments of coding DNA interspersed with long stretches of non-coding (intron) DNA, and the near ubiquity of sexual reproduction. As is well known, alternative splicing of its coding sequences enables one transcription unit to produce multiple variants of each encoded protein. Additionally, padding transcription units with non-coding DNA (often many thousands of base pairs long) provides a readily evolvable way to set how soon in a cell cycle the various mRNAs will begin being expressed and the total amount of mRNA that each transcription unit can make during a cell cycle. This regulation complements control via the transcriptional promoter and facilitates the creation of complex eukaryotic cell types, tissues, and organisms. However, it also makes eukaryotes exceedingly vulnerable to double-strand DNA breaks, which end-joining break repair pathways can repair incorrectly. Transcription units cover such a large fraction of the genome that any mis-repair producing a reorganized chromosome has a high probability of destroying a gene. During meiosis, the synaptonemal complex aligns homologous chromosome pairs and the pachytene checkpoint detects, selectively arrests, and in many organisms actively destroys gamete-producing cells with chromosomes that cannot adequately synapse; this creates a filter favoring transmission to the next generation of chromosomes that retain the parental organization, while selectively culling those with interrupted transcription units. This same meiotic checkpoint, reacting to accidental chromosomal reorganizations inflicted by error-prone break repair, can, as a side effect, provide a mechanism for the formation of new species in sympatry. It has been a long-standing puzzle how something as seemingly maladaptive as hybrid sterility between such new species can arise. I suggest that this paradox is resolved by understanding the adaptive importance of the pachytene checkpoint, as outlined above.

SYNOPSIS: Cet essai vise à expliquer deux énigmes biologiques : pourquoi les unités de transcription eucaryotes sont composées de courts segments d'ADN codant entrecoupés de longues portions d'ADN non codant (intron) et la quasi-omniprésence de la reproduction sexuée. Comme nous le savons, l'épissage alternatif des séquences codantes permet à une unité de transcription de produire de multiple variant de chacune des protéines codées. De plus, remplir les unités de transcription avec de l'ADN non codant (souvent plusieurs milliers de paires de bases) fournit un moyen facilement évolutif de définir à quel moment dans un cycle cellulaire les différents ARNm commenceront à être exprimés et quelle quantité totale d'ARNm sera produite par chaque unité de transcription au cours d'un cycle cellulaire. Cette régulation s'ajoute au contrôle par le promoteur transcriptionnel et facilite la création de types cellulaires eucaryotes complexes, de tissus et d'organismes. Cependant, cela rend également les eucaryotes extrêmement vulnérables aux cassures double brin de l'ADN, que les voies de réparation par jonction des extrémités non-homologues peuvent réparer de manière inexacte. Les unités de transcription couvrent une fraction si importante du génome que toute mauvaise réparation produisant un chromosome réorganisé a une forte probabilité de détruire un gène. Au cours de la méiose, le complexe synaptonémal aligne les paires de chromosomes homologues et le point de contrôle du pachytène détecte, arrête sélectivement et dans de nombreux organismes détruit activement les cellules productrices de gamètes possédant des chromosomes qui ne peuvent pas s'apparier correctement. Cela crée un filtre favorisant la transmission à la génération suivante de chromosomes conservant l'organisation parentale, tout en éliminant sélectivement ceux dont les unités de transcription ont été interrompues. Ce même point de contrôle méiotique, réagissant aux réorganisations chromosomiques accidentelles résultantes d'erreurs lors de la réparation des cassures double-brin, peut, comme effet secondaire, fournir un mécanisme d'émergence de nouvelles espèces sympatriques. La question de comprendre comment quelque chose d'aussi apparemment inadapté que la stérilité hybride entre ces nouvelles espèces peut survenir reste un casse-tête de longue date. Je suggère que ce paradoxe soit résolu en comprenant l'importance adaptative du point de contrôle du pachytène, comme indiqué ci-dessus.

Rsumen: Este ensayo tiene como objetivo explicar dos enigmas biológicos: por qué las unidades de transcripción eucarióticas están compuestas de segmentos cortos de ADN codificante intercalados con largos tramos de ADN no codificante (intrones) y la práctica ubicuidad de la reproducción sexual. Como es bien sabido, el corte y empalme alternativo de sus secuencias codificantes permite que una unidad de transcripción produzca múltiples variantes de cada proteína codificada. Además, el relleno de unidades de transcripción con ADN no codificante (a menudo de muchos miles de pares de bases de largo) proporciona un mecanismo evolutivo sencillo para establecer con cuánta rapidez los diversos ARNm comenzarán a expresarse y la cantidad total de ARNm que cada unidad de transcripción puede generar durante un ciclo celular. Esta regulación complementa al control a través del promotor transcripcional y facilita la creación de tipos celulares, tejidos y organismos eucariotas complejos. Sin embargo, también hace que los eucariotas sean extremadamente vulnerables a las roturas de ADN de doble cadena, que pueden ser reparadas incorrectamente por las vías de reparación de roturas de unión de extremos. Las unidades de transcripción cubren una fracción tan grande del genoma que cualquier reparación incorrecta que produzca un cromosoma reorganizado tiene una alta probabilidad de destruir un gen. Durante la meiosis, el complejo sinaptonémico alinea pares de cromosomas homólogos y el punto de control de paquitena detecta, detiene selectivamente y, en muchos organismos, destruye activamente las células productoras de gametos con cromosomas que no pueden hacer sinapsis de manera adecuada; esto crea un filtro que favorece la transmisión a la siguiente generación de cromosomas que retienen la organización parental, al tiempo que elimina selectivamente aquellos con unidades de transcripción interrumpidas. Este mismo punto de control meiótico, que reacciona a las reorganizaciones cromosómicas accidentales infligidas por la reparación de roturas propensa a errores, puede, como efecto secundario, proporcionar un mecanismo para la formación de nuevas especies en simpatría. Durante mucho tiempo, ha sido un enigma cómo puede surgir algo tan aparentemente inadaptado como la esterilidad híbrida entre estas nuevas especies. Propongo que esta paradoja se resuelva comprendiendo la importancia adaptativa del punto de control de paquitena, como se describió anteriormente.

Resumo: Este ensaio visa explicar dois enigmas biológicos: o porquê das unidades de transcrição eucarióticas serem compostas por segmentos curtos de DNA codificante intercalados por longos trechos de DNA não-codificante (íntron), e a quase universalidade da reprodução sexual. Como é bem conhecido, o splicing alternativo de sequências codificantes permite que uma unidade de transcrição produza múltiplas variantes de cada proteína codificada. Além disso, o preenchimento de unidades de transcrição com DNA não-codificante (geralmente muitos milhares de pares de bases) fornece uma maneira pronta para evoluir e determinar o quão cedo no ciclo celular os diversos mRNAs começarão a ser expressos e a quantidade total de mRNA que cada unidade de transcrição irá produzir durante um ciclo celular. Esta regulação complementa o controle através do promotor transcricional e facilita a geração de tipos complexos de células eucarióticas, tecidos e organismos. No entanto, também torna os eucariotos extremamente vulneráveis a quebras de DNA de fita dupla, dado que que os mecanismos de reparo da quebra da fita dupla podem reparar incorretamente. As unidades de transcrição cobrem uma fração tão grande do genoma que qualquer reparo incorreto que produza um cromossomo reorganizado tem uma alta probabilidade de quebrar um gene. Durante a meiose, o complexo sinaptonêmico alinha pares de cromossomos homólogos e o ponto de verificação do paquíteno detecta, interrompe seletivamente e, em muitos organismos, destrói ativamente células produtoras de gametas com cromossomos que não podem fazer sinapse adequadamente; isso cria um filtro que favorece a transmissão de cromossomos que retêm a organização parental para a próxima geração, enquanto seleciona seletivamente aqueles com unidades de transcrição interrompidas. Esse ponto de verificação meiótico, que responde a reorganizações cromossômicas acidentais infligidas por reparos de quebras propensos a erros, pode, como efeito colateral, também ser um mecanismo de formação de novas espécies em simpatria. O enigma de como algo aparentemente com tão baixo valor adaptativo quanto a esterilidade híbrida entre essas novas espécies pode surgir permanece há muito tempo. Eu proponho que esse paradoxo seja resolvido pela compreensão da importância adaptativa do ponto de verificação do paquíteno, conforme descrito acima.

Centralspindlin and chromosomal passenger complex behavior during normal and Rappaport furrow specification in echinoderm embryos.

The chromosomal passenger (CPC) and Centralspindlin complexes are essential for organizing the anaphase central spindle and providing cues that position the cytokinetic furrow between daughter nuclei. However, echinoderm zygotes are also capable of forming "Rappaport furrows" between asters positioned back-to-back without intervening chromosomes. To understand how these complexes contribute to normal and Rappaport furrow formation, we studied the localization patterns of Survivin and mitotic-kinesin-like-protein1 (MKLP1), members respectively of the CPC and the Centralspindlin complex, and the effect of CPC inhibition on cleavage in mono- and binucleate echinoderm zygotes. In zygotes, Survivin initially localized to metaphase chromosomes, upon anaphase onset relocalized to the central spindle and then, together with MKLP1 spread towards the equatorial cortex in an Aurora-dependent manner. Inhibition of Aurora kinase activity resulted in disruption of central spindle organization and furrow regression, although astral microtubule elongation and furrow initiation were normal. In binucleate cells containing two parallel spindles MKLP1 and Survivin localized to the plane of the former metaphase plate, but were not observed in the secondary cleavage plane formed between unrelated spindle poles, except when chromosomes were abnormally present there. However, the secondary furrow was sensitive to Aurora inhibition, indicating that Aurora kinase may still contribute to furrow ingression without chromosomes nearby. Our results provide insights that reconcile classic micromanipulation studies with current molecular understanding of furrow specification in animal cells.

An agent-based model contrasts opposite effects of dynamic and stable microtubules on cleavage furrow positioning.

From experiments by Foe and von Dassow (Foe, V.E., and G. von Dassow. 2008. J. Cell Biol. 183:457-470) and others, we infer a molecular mechanism for positioning the cleavage furrow during cytokinesis. Computer simulations reveal how this mechanism depends on quantitative motor-behavior details and explore how robustly this mechanism succeeds across a range of cell sizes. The mechanism involves the MKLP1 (kinesin-6) component of centralspindlin binding to and walking along microtubules to stimulate cortical contractility where the centralspindlin complex concentrates. The majority of astral microtubules are dynamically unstable. They bind most MKLP1 and suppress cortical Rho/myosin II activation because the tips of unstable microtubules usually depolymerize before MKLP1s reach the cortex. A subset of astral microtubules stabilizes during anaphase, becoming effective rails along which MKLP1 can actually reach the cortex. Because stabilized microtubules aim statistically at the equatorial spindle midplane, that is where centralspindlin accumulates to stimulate furrow formation.

Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation.

The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine19-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation. With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase; among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis.