Module-based subnetwork alignments reveal novel transcriptional regulators in malaria parasite Plasmodium falciparum

Background Malaria causes over one million deaths annually, posing an enormous health and economic burden in endemic regions. The completion of genome sequencing of the causative agents, a group of parasites in the genus Plasmodium, revealed potential drug and vaccine candidates. However, genomics-driven target discovery has been significantly hampered by our limited knowledge of the cellular networks associated with parasite development and pathogenesis. In this paper, we propose an approach based on aligning neighborhood PPI subnetworks across species to identify network components in the malaria parasite P. falciparum. Results Instead of only relying on sequence similarities to detect functional orthologs, our approach measures the conservation between the neighborhood subnetworks in protein-protein interaction (PPI) networks in two species, P. falciparum and E. coli. 1,082 P. falciparum proteins were predicted as functional orthologs of known transcriptional regulators in the E. coli network, including general transcriptional regulators, parasite-specific transcriptional regulators in the ApiAP2 protein family, and other potential regulatory proteins. They are implicated in a variety of cellular processes involving chromatin remodeling, genome integrity, secretion, invasion, protein processing, and metabolism. Conclusions In this proof-of-concept study, we demonstrate that a subnetwork alignment approach can reveal previously uncharacterized members of the subnetworks, which opens new opportunities to identify potential therapeutic targets and provide new insights into parasite biology, pathogenesis and virulence. This approach can be extended to other systems, especially those with poor genome annotation and a paucity of knowledge about cellular networks

Systems Biology Approaches Towards Immunity against <em>Plasmodium</em>

Malaria is one of the most devastating infectious diseases known to humans. It is caused by unicellular protozoan parasites belonging to the genus Plasmodium. Till date, over 200 species of Plasmodium have been formally described, and each species infects a certain range of hosts. However, the human infection is limited to only five of the species, of which P. falciparum is the most responsible. Due to the emergence of parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides which threaten the control programmes, new antimalarial therapeutics or approaches capable of predicting useful models of how different cells of the innate immune system function, is the need of the hour. Systems Immunology is a relatively recent discipline under Systems Biology to understand the structure and function of the immune system and how the components of the immune system work together as a whole. Thus, this chapter aims to give insight into the approaches of Systems Biology for investigating the immune factors that are formed during Plasmodium falciparum infection in the human body. Here, the numerous experimental and computational works with the ongoing methodologies using Systems Biology approaches along with the interactions of host and pathogen will be discussed

Evidence-Based Annotation of the Malaria Parasite's Genome Using Comparative Expression Profiling

A fundamental problem in systems biology and whole genome sequence analysis is how to infer functions for the many uncharacterized proteins that are identified, whether they are conserved across organisms of different phyla or are phylum-specific. This problem is especially acute in pathogens, such as malaria parasites, where genetic and biochemical investigations are likely to be more difficult. Here we perform comparative expression analysis on Plasmodium parasite life cycle data derived from P. falciparum blood, sporozoite, zygote and ookinete stages, and P. yoelii mosquito oocyst and salivary gland sporozoites, blood and liver stages and show that type II fatty acid biosynthesis genes are upregulated in liver and insect stages relative to asexual blood stages. We also show that some universally uncharacterized genes with orthologs in Plasmodium species, Saccharomyces cerevisiae and humans show coordinated transcription patterns in large collections of human and yeast expression data and that the function of the uncharacterized genes can sometimes be predicted based on the expression patterns across these diverse organisms. We also use a comprehensive and unbiased literature mining method to predict which uncharacterized parasite-specific genes are likely to have roles in processes such as gliding motility, host-cell interactions, sporozoite stage, or rhoptry function. These analyses, together with protein-protein interaction data, provide probabilistic models that predict the function of 926 uncharacterized malaria genes and also suggest that malaria parasites may provide a simple model system for the study of some human processes. These data also provide a foundation for further studies of transcriptional regulation in malaria parasites

Visualising plasmodium falciparum functional genomic data in MaGnET: malaria genome exploration tool

Malaria affects the lives of 500 million people around the world each year. The disease is caused by protozoan parasites of the genus Plasmodium, whose ability to evade the immune system and quickly evolve resistance to drugs poses a major challenge for disease control. The results of several Plasmodium genome sequencing projects have revealed how little is known about the function of their genes (over half of the approximately 5400 genes in Plasmodium falciparum, the most deadly human parasite, are annotated as hypothetical ). Recently, several large-scale studies have attempted to shed light on the processes in which genes are involved; for example, the use of DNA microarrays to profile the parasite s gene expression. With the emergence of varied types of functional genomic data comes a need for effective tools that allow biologists (and bioinformaticians) to explore these data. The goal of exploration/browsing-style analyses will typically be to derive clues towards the function of thus far uncharacterised gene products, and to formulate experimentally testable hypotheses. Graphic interfaces to individual data sets are obviously beneficial in this endeavour. However, effective visual data exploration requires also that interfaces to different functional genomic data are integrated and that the user can carry forward a selected group of genes (not merely one at a time) across a variety of data sets. Non-expert users especially benefit from workbenchlike tools offering access to the data in this way. Still, only very few of the contemporary publicly available software have implemented such functionality. This work introduces a novel software tool for the integrated visualisation of functional genomic data relating to P. falciparum: the Malaria Genome Exploration Tool (MaGnET). MaGnET consists of a light-weight Java program for effective visualisation linked to a MySQL database for data storage. In order to maximise accessibility, the program is publicly available over the World Wide Web (http://www.malariagenomeexplorer.org/). MaGnET incorporates a Genome Viewer for visualising the location of genomic features, a Protein-Protein Interaction Viewer for visualising networks of experimentally determined interactions and an Expression Data Viewer for displaying mRNA and protein expression data. Complex database queries can easily be constructed in the Data Analysis Viewer. An advantage over most other tools is that all sections are fully integrated, allowing users to carry selected groups of genes across different datasets. Furthermore, MaGnET provides useful advanced visualisation features, including mapping of expression data onto genomic location or protein-protein interaction network. The inclusion of available third-party Java software has expanded the visualisation capability of MaGnET; for example, the Jmol viewer has been incorporated for viewing 3-D protein structures. An effort has been made to only include data in MaGnET that is at least of reasonable quality. The MaGnET database collates experimental data from various public Plasmodium resources (e.g. PlasmoDB) and from published functional genomic studies, such as DNA microarrays. In addition, through careful filtering and labelling we have been able to include some predicted annotation that has not been experimentally confirmed, such as Gene Ontology and InterPro functional assignments and modelled protein structures. The application of MaGnET to malaria biology is demonstrated through a series of small studies. Initial examples show how MaGnET can be used to effectively demonstrate results from previously published analyses. This is followed up by using MaGnET to make a set of predictions about the possible functions of selected uncharacterised genes and suggesting follow-up experiments

Proteômica funcional da calmodulina na diferenciação dos merozoítos e na invasão eritrocitária de Plasmodium falciparum

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2018.INTRODUÇÃO: Malária ainda é considerada um grande desafio para a saúde pública; e foi responsável por aproximadamente 438.000 mortes em 2015. Plasmodium falciparum transmite a forma mais complicada da malária em humanos, a malária cerebral. O ciclo intraeritrocitário é o responsável pelo crescimento exponencial da infecção no hospedeiro vertebrado e, consequentemente, pelos sintomas clínicos da doença, o que torna essa etapa um interessante alvo para drogas de combate à doença. Calmodulina (CaM) é um sensor de cálcio ubíquo, importante para sinalização celular. A interação entre CaM e suas parceiras (CBPs) podem ser também essenciais para os processos em P. falciparum, o que torna essas interações interessantes alvos terapêuticos. OBJETIVOS: combinar estratégias bioinformáticas e experimentais para estudo da interação entre PfCaM e suas parceiras, para elucidar novos alvos para o combate à malária. MÉTODOS: Foi realizada um screening virtual baseado no princípio de cross-reference species, visando encontrar parceiros de PfCaM ao estudar suas interações em outras espécies. PfCaM foi expressa heterologamente e purificada por cromatografia do tipo IMAC, eluída com 100mM de imidazol. A cultura de P. falciparum foi enriquecida na fase esquizonte para se estudar as interações feitas por PfCaM, e essa amostra foi usada para ensaios de cromatografia de afinidade à calmodulina e BN-PAGE. RESULTADOS: O screening virtual teve como melhor resultado de CBP para posterior análise a proteína Hsp70-x, a sequência contém um peptídeo conservado na família Hsp70 descrito como ligante à CaM. A expressão heteróloga e purificação de PfCaM foi bem-sucedida, e a proteína está apta para posteriores ensaios de PPI. A interação entre PfCaM e Hsp70-x foi encontrada na cromatografia de afinidade; além disso, interações entre PfCaM e importantes moléculas efetoras que foram encontradas na cromatografia de afinidade foram discutidas. A cromatografia de afinidade à calmodulina mostrou-se uma estratégia interessante para se estudar os parceiros de PfCaM, além de colocar PfCaM como potencial regulador de importantes moléculas efetoras envolvidas na invasão de novas hemácias. A abordagem BN-PAGE foi estabelecida para estudar os complexos em P. falciparum. CONCLUSÕES: a combinação de estratégias bioinformáticas e experimentais mostrou-se uma boa estratégia para se elucidar interações entre proteínas em P. falciparum. Espera-se com esse trabalho ter destacado importantes interações com potencial para alvos terapêuticos visando o combate da malária.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), Fundação de Amparo à Pesquisa do Distrito Federal (FAPDF), Financiadora de Estudos e Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Comité Français d'Évaluation de la Coopération Universitaire et Scientifique avec le Brésil (COFECUB).BACKGROUND: Malaria is still a big challenge for public health; it was responsible for about 438.000 deaths in 2015. Plasmodium falciparum transmits cerebral malaria, the most severe complication related to a Plasmodium spp. infection in humans. The asexual intraerythrocytic cycle is responsible for increasing infection and, consequently, for clinical symptoms of malaria, reason why it is a target for drug development. Calmodulin (CaM) is a ubiquitous calcium sensor, responsible for important cell signaling processes. Since its structure is conserved among eukaryotes, CaM-binding proteins (CBPs) can be also essential for these processes in Plasmodium spp and, for consequence, good drug targets. AIM: to combine bioinformatical and experimental approaches aiming at studying the interaction between PfCaM and its partners, to elucidate new drug targets to fight against malaria. METHODS: a virtual screening based on cross-reference species information was made, to predict CBPs in P. falciparum. PfCaM was heterologously expressed and purified by IMAC chromatography, eluted with 100mM of imidazole. A P. falciparum cellular culture was enriched in schizont phase, and the final sample was used to perform CaM-affinity chromatography and BN-PAGE approaches; RESULTS: The virtual screening has predicted P. falciparum’s HSP70x as a potential CBP; PfHsp70-x sequence contains calmodulin-binding peptides already described in literature. Heterologous PfCaM was successfully expressed and purified and can perform further PPI assays. It was found an indicative of interaction between PfCaM and PfHsp 70-x by CaM-affinity chromatography; besides, interactions between PfCaM and important molecular effectors involved in invasion of new cells were discussed. Chromatography approach has shown to be an interesting methodology to infer P. falciparum calmodulin-binding partners and pointed PfCaM as a potential regulator of important molecular effectors involved in erythrocyte invasion. BN-PAGE approach was established to study P. falciparum protein complexes. CONCLUSIONS: Combined experimental and in silico approaches seem to be a good strategy to evaluate protein-protein interactions. At the end of this work, we aim at having good potential drug targets for further studies in malaria disease

'Schistosoma mansoni' : integration of phosphoproteomics, kinomics and CaMKII

'Schistosoma mansoni' is a parasitic trematode and the causative agent of the debilitating disease human schistosomiasis. This study focused on global signalling processes which occur in the parasite and on Ca2+/Calmodulin Kinase II (CaMKII) signalling. Phosphoproteomics of a mixed population of adult worms was performed, identifying a total of 15,844 phosphopeptides, from 3,176 proteins with 12,936 unique phosphorylation sites. Analysis of the phosphorylated sites/proteins provided valuable insights into the complexities of signalling in this parasite, including insights into overrepresented and novel phosphorylation site motifs, putative upstream protein kinases, molecular function, and interactions between phosphoproteins (1,586 nodes and 12,733 edges). Highly connected interaction clusters highlighted the pivotal role of phosphorylated ribosomal and ribosomal biogenesis proteins, proteasome, and spliceosome proteins within S. mansoni. Furthermore, analysis of the protein kinases revealed 808 phosphorylation sites amongst 136 protein kinases representing 54% of the S. mansoni kinome. This novel, deep, phosphoproteomic dataset supported the development of the first parasite-specific kinomic array comprising 96 peptides with many upstream kinases predicted. The array was deployed to identify eight peptides (proteins) which were significantly differentially phosphorylated by homogenates from male and female adult worms: the serine/threonine protein kinases CDK/PITSLRE, AMPK, AKT, SmTK4, insulin receptor, protein phosphatase 2c gamma, Rho2 GTPase, and vacuolar protein sorting 26. CaMKII was identified as a protein of interest in the phosphoproteomic analysis, further investigations were carried out. Using anti-phospho-CaMKII antibodies, CaMKII was detected at approximately 57 kDa in cercariae, somules, and adult worms. Activated CaMKII was located visually in the nervous system, excretory system, sensory structures, and the tegument as well as in the female vitellaria. Inhibition assays determined that CaMKII phosphorylation (activation) in somules could be suppressed by KN93, and further assays found that the anti-schistosomal drug praziquantel had a significant effect on CaMKII activation in S. mansoni. Finally, suppression of CaMKII expression by small interfering RNA (siRNA) resulted in a 66% and 52% reduction in males and females respectively. Collectively these studies show that phosphorylation plays a wide reaching and crucial role in the complex signalling pathways that ultimately control S. mansoni from highlighting their involvement in reproduction and proliferation, host location and movement to commanding cell death, the elucidation of these phosphorylating pathways are vital to understanding this parasites biology

Cryptic reservoirs of micro-eukaryotic parasites in ecologically relevant intertidal invertebrates from temperate coastal ecosystems unveiled by a combined histopathological, ultrastructural, and molecular approach

271 p.La mayoría de los eucariotas son organismos unicelulares (protistas), muchos de ellos pertenecientes a linajes que divergieron temprano en la historia evolutiva de este Dominio de organismos nucleados. Microscópicos, enormemente diversos y fenotípicamente convergentes, su clasificación cladística ha sido históricamente compleja, dejando atrás un extenso registro de taxones y de términos parafiléticos y polifiléticos. Teniendo que investigar atributos estructurales, celulares, biológicos y ecológicos en un mundo de rápidas interacciones y difícilmente accesible a simple vista, la protistología es particularmente dependiente de la sistemática. Ésta permite inferir rasgos de especies crípticas a partir de especies evolutivamente relacionadas.Las moléculas de ADN (y ARN), representan un "registro" preciso de estos eventos de diversificación, que preceden incluso a los más antiguos registros fósiles. En los últimos años, la maduración de los métodos de filogenia molecular, catalizados por una mayor accesibilidad a la secuenciación de próxima generación (NGS), está permitiendo resolver preguntas e hipótesis sobre la evolución y la especiación de estos organismos micro-eucariotas que no se habían podido responder mediante otros métodos. Por una parte, arboles filogenéticos construidos mediante concatenaciones de cientos, incluso miles de genes, están permitiendo rastrear la historia evolutiva de los linajes protistas hasta el último ancestro común de todos los eucariotas (LECA). Concomitantemente, análisis moleculares basados en genes e incluso fragmentos cortos (especialmente 18S rRNA), recuperados principalmente de matrices ambientales u orgánicas (eDNA o ADN ambiental), están revelando una ¿caja de Pandora¿ de diversidad micro-eucariota. Ésta diversidad ¿oculta¿ está transformando nuestra percepción de los protistas en la cadena trófica y la estructura ecológica. En el medio marino, sus papeles como autótrofos, heterótrofos (predadores, saprófitos, parásitos) o mixótrofos crece en importancia día a día. El aumento simultáneo de diversidad e importancia ha sido particularmente pronunciado entre los linajes de parásitos protistas, que adaptados a la vida dentro de un huésped son más inaccesibles y morfológicamente indistinguibles que sus homólogos de vida libre. Muy competitivo como estilo de vida, el parasitismo ha evolucionado de forma independiente varias veces en prácticamente todos los grupos eucariotas, en algunos incluso cientos de veces. De hecho, es posible que el efecto parapátrico que implica una existencia endosimbiótica, haya exacerbado la especiación entre los parásitos, que representan la que posiblemente sea la más común estrategia de consumo entre los organismos vivos. Es más, el número de especies crípticas que están, a día de hoy, siendo descubiertas en la mayoría de los linajes de parásitos protistas sigue aumentando abruptamente o apenas comienza a mostrar una desaceleración. Cabe destacar, que el descubrimiento de esta diversidad oculta, incluidas las especies crípticas, va más allá de la escalada en el número de especies; afecta los estudios sobre biología celular, ciclos biológicos, y ecología. Inexorablemente, esta fuerza mostrada por los métodos de análisis y secuenciación del ADN está abriendo una brecha entre la diversidad genética existente y nuestra comprensión de la morfología, patología, transmisión y posibles hospedadores de los parásitos protistas que la constituyen. Este desequilibrio es particularmente evidente entre los parásitos que infectan linajes de invertebrados, los cuales, salvo algunos taxones con interés comercial, permanecen en gran parte sin analizar, a pesar de constituir un grupo mucho más diverso que los vertebrados. Por una parte, es lógico que los parásitos protistas causantes de infecciones en especies marinas de interés comercial (peces, bivalvos, crustáceos¿) hayan sido priorizadas, pero hay que tener en cuenta que muchos de estos micro-eucariotas tienen ciclos de vida complejos, en los que pequeños invertebrados actúan muchas veces como vectores o reservorios. Descubrir y contextualizar estas asociaciones puede ser determinante a la hora de comprender cuándo y dónde puede variar la presión y capacidad infectiva de algunas de estas infecciones en la comunidad o huéspedes específicos. Al mismo tiempo que su diversidad e importancia aumenta, la inclusión progresiva de parásitos en modelos ecológicos está experimentando variaciones de gran alcance en la dinámica poblacional de las especies animales, vegetales o fúngicas en los ecosistemas. En consecuencia, las asociaciones entre parásitos y hospedadores se investigan cada vez más como una parte importante de la estructura de la comunidad, y no exclusivamente como una "molestia" para el ser humano y sus intereses. Por desgracia la inclusión de parásitos en modelos ecológicos está siendo lastrada por un profundo desconocimiento de estas interacciones. A diferencia de los organismos multicelulares, que han podido ser observados por científicos y aficionados durante siglos, la distribución espaciotemporal de la mayoría de los organismos unicelulares sigue siendo un profundo misterio. No obstante, dadas sus importantes funciones como vectores, huéspedes intermediarios y reservorios, una comprensión mucho más profunda del patobioma (patógenos asociados a un hospedador) y su variabilidad espacio-temporal es de suma importancia para un mayor poder de predicción de los factores de presión causantes de epidemias o zoonosis en el huésped, la población y el medioambiente.En este contexto, la hipótesis de este estudio plantea que especies de invertebrados comunes en la zona inter-mareal de ecosistemas costeros en climas templados son reservorios crípticos de un número significativo de parásitos micro-eucariotas (protistas) de interés para el medio y los recursos marinos. Eldescubrimiento progresivo de estas asociaciones ocultas de parásitos-huéspedes (mediante exámenes combinando técnicas histopatológicas, ultraestructurales y moleculares) permite una mejor comprensión de la morfología, patología, biología celular y ciclo de vida de dichos patógenos, lo que a su vez consiente un seguimiento más estrecho de los factores y presiones que promueven epidemias y zoonosis en una escala espacio-temporal.PIE:Plentziako Itsas Estazio

The evolution of protein kinase specificity

All research conducted at EMBL-EBI under the supervision of Dr. Pedro Beltrao. Work on the PhD project was paused temporarily in the Spring of 2017 for me to undertake a 3-month internship at EMBO Press (in Heidelberg).Protein phosphorylation represents one of the most important post-translational modifica- tions (PTMs) for cell signalling, and is is catalysed by a group of enzymes called protein kinases. Through this activity they serve as key regulators of almost all cellular processes. This is achieved at any time by a network of different kinases that are transiently active. The fidelity of cell systems control therefore requires that each kinase targets only a restricted set of substrates. This specificity is achieved partly by contextual factors that separate kinases spatially and temporally, but also by sequence features that are encoded in the kinase domain itself. For this thesis I focus on elements of kinase specificity that are encoded in the the active site of the enzyme. During these investigations I have tried to address three main questions: 1) How is specificity for residues surrounding the phosphorylation site determined in the kinase? 2) How did these specificities evolve? and 3) To what extent does kinase evolution correlate with the evolution of its substrates? First, I developed a sequence-based method for the automated detection of kinase speci- ficity determining residues (SDRs). The putative determinants were then rationalised using available structural data, and in two specific cases were validated experimentally. I also used mutation data from The Cancer Genome Atlas (TCGA) to demonstrate that kinase SDRs are often targeted during cancer. Second, a global analysis of SDR evolution was performed for kinases following gene duplication and speciation, revealing that SDRs often diverge between paralogues but not between orthologues. This global analysis is followed by a detailed case study of G-protein coupled receptor kinase (GRKs) evolution using ancestral sequence reconstructions. Third, I inferred global substrate preferences in a taxonomically broad range of species using phosphoproteome data. I then related the evolution of substrate motif sequences to that of their cognate effector kinases where possible. The results strongly suggest that many of the motifs emerged in a universal eukaryotic ancestor. I finish by summarising the major findings of this doctoral research, which to my knowl- edge represents the most comprehensive analysis to date of protein kinase specificity and its evolution.BBSR

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin