Cell biology and Biotechnology

Mitotic spindle nucleation and coordination with the cell cycle

Dr Victor Álvarez Tallada
Researcher associated to Dr Juan Jiménez Martínez

Summary
Five relevant publications
Lab members & collaborators

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Summary

We are working in two main research lines in the Lab 126:

1.- Activation and regulation of the early stages of interphase and mitotic microtubule assembly.

Numerous biological functions in interphase and the equal segregation of genetic information between the two daughter cells generated by mitosis, or between the four gametes generated by meiosis, depend dramatically on the function of microtubule bundles. In interphase, microtubules have morphological and transport functions, among others. In mitosis and meiosis they physically trap chromosome masses and separate them to opposite poles of the cell. Microtubules polymerise from protein complexes called MTOCS (MicroTubule Organising Centres) in interphase and Centrosomes in mitosis and meiosis. Effective nucleation occurs from the ɣ -Tubulin (ɣ -TuSC) subcomplex, which is evolutionarily highly conserved in eukaryotes from yeast to humans. However, much of the molecular mechanisms by which ɣ -TuSC activates or deactivates microtubule nucleation, and how this process coordinates with the cell cycle or responds to situations that alter normal cell physiology, are not yet well characterised. However, it is well documented that mutations in many of these genes that alter spindle function lead to aberrant separations of genetic material (aneuploidy) which are the molecular cause of several types of tumours. In our lab we use the fission yeast S. pombe as a model system to investigate the molecular signals that activate and modulate the intensity of microtubule nucleation. This organism, widely used in research, is an extremely versatile experimental system. It allows us to combine powerful techniques from Genetics, Cell and Molecular Biology, high-speed confocal microscopy in living cells, Biochemistry and state-of-the-art "omics" analyses to investigate the molecular mechanisms that govern microtubule nucleation function and to characterise the action of new elements involved in its regulation.
In our group we have mutants such as the one in image 1 in which only one of the centrosomes is activated (above panels: wild-type cell, below an example of a mutant cell); or the one in image 2 in which prophase, which normally takes 2 minutes, is prolonged up to 40 minutes without activation of the nucleating centres. These and other mutants we handle in our lab are excellent genetic backgrounds to ask what factors are necessary to activate microtubule synthesis.
 




2.- Functional relationship between RNA processing factors for the maintenance of genomic stability.

The correct duplication and segregation of chromosomes is essential for the viability of any eukaryotic cell. The accumulation of mutations and rearrangements or the asymmetric distribution of genetic material during the mitotic process leads to cellular defects known generically as genomic instability. This is a common feature in the ageing process, in various congenital diseases and especially in different types of cancer. Chromosomal rearrangements and asymmetric inheritance in chromosome number (aneuploidy) influence the abnormalities of subsequent divisions and are responsible for cellular heterogeneity within the tumour itself. This intra-tumoural genomic heterogeneity is what determines such important aspects in the evolution of the oncogenic process as the development of metastasis and resistance to therapy. In our laboratory we have identified a new conditional mutation responsible for an extreme phenotype of genomic instability (image 3).



This mutation resides in an essential gene (its complete deletion is lethal) and universally conserved in eukaryotes. The affected gene (Cwf15/CWC15/HSPC148) is curiously annotated as a splicing factor, involved in RNA processing. Although nothing is known about its molecular function, this protein has been identified as a tumour antigen in the bladder cancer cell line BLZ211. In our experiments, we discovered that this mutation in the cwf15 gene (cwf15-d53) generates two very marked deleterious phenotypes: Firstly, at the molecular level, we observed a very significant increase in the transcription of intergenic and antisense regions that are absent in the wild-type strain. This phenotype (known as transcription readthrough) is normally caused by dysregulation of RNA polymerase II at the end of transcription. Consistently, the cwf15-d53 mutation causes synthetic lethality when combined with the lack of a transcription termination factor (Rhn1), which alone is viable. Furthermore, deletion of the rhn1 factor under the same restrictive conditions for the cwf15-d53 mutation, mimics the cwf15 lack-of-function phenotype. On the other hand, interestingly enough, a dominant mutation in another essential termination factor (Yth1) renders the function of cwf15 completely dispensable. Therefore, in addition to the function associated with splicing (excision of intronic, non-coding, sequences of messenger RNAs), our data suggest that this gene has an important and novel function in the regulation of the end of transcription. Second, at the cellular level, we observed an aberrant distribution of loaded cohesin onto chromosomes, both quantitatively and qualitatively. Although there are indications that these two phenotypes may be related, the molecular mechanism that may connect them is not known in detail.

The objectives of this research project are therefore to determine the function of this protein in the regulation of the end of transcription, as well as the molecular mechanism that may connect this novel function with the dynamics of cohesin, whose alteration seems to be the cause of the observed genomic instability. The results may shed light on new essential functions in the maintenance of genomic stability and identify as yet unknown molecular targets for the development of new treatments for diseases with this genetic basis. We are also currently trying to demonstrate the role of this gene in the development and evolutionary conservation of the processes we are studying between S. pombe and D. melanogaster in collaboration with Dr James Castelli's group.

Why fission yeast as a model?

Our biological model, Schizosaccharomyces pombe, is a single-celled ascomycete fungus with a cylindrical shape. It is commonly known as fission yeast because it grows in interphase at the tips of the cell and in mitosis divides by fission in the central part, generating two equal sized daughter cells. This yeast has been used as a model for numerous biological processes since 1950 because of the possibility it offers, in comparison to other models, to simplify the study of very complex processes. One of the many examples is illustrated by the Discovery, for the first time, of the main factors that regulate the progress, transitions and checkpoints of the cell cycle; processes common to all eukaryotic cells, from yeast to human. In fact, due to the interest in this yeast, the S. pombe genome was the sixth eukaryotic genome to be completely sequenced.
The experimental power of this yeast lies in aspects that together make it stand out from other models. It has a generation time of between 2.5 and 4 hours (in one night we can have great-great-granddaughters); its culture is very simple and inexpensive; it transforms very easily with exogenous DNA and its homologous recombination systems allow targeted integrations in the genome that enable highly potent gene editing; There are available in the community a multitude of plasmids, constitutive and regulatory promoters and selection markers; tens of thousands of constitutive and conditional mutants, as well as fluorescent protein tags for live cell microscopy that allow elaborate dynamic studies. But genetic versatility is undoubtedly the most distinguishing feature. We can propagate it in a haploid or diploid cycle, which allows us to study recessive mutations directly or to maintain deleterious mutations in heterozygosis. We can cross two haploid strains of different mating types and to separate and analyse individually the 4 gametes (spores) coming from individual meiosis. This allows us to obtain multiple mutant combinations very quickly and efficiently. In fact, the automation of all these processes has generated massive studies of drug sensitivity or genetic interactions between mutants (known as High Throughput Screens or HTS) that continue to provide tremendously valuable information about the functioning of a living cell.
 

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