Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of alpha tubulin 1
Protein (function)
Microtubules are cytoskeletal filaments formed as hollow cylinders from dimers of a-tubulin and b-tubulin. Eukaryotic cells can arrange microtubules into different assemblies, including those forming axonemes of flagella and cilia, spindles for genome segregation during cell division, or cytoplasmic asters originating from the microtubule organizing centre and mediating intracellular transport and force distribution.
In some organisms, different isoforms of tubulin are expressed in different cells or tissues, suggesting that the different physical and biological properties of microtubules can be encoded in the subtle variations of the protein sequence of these isoforms.
Work on spindle microtubules shows that their number depends on the numbers of kinetochores. Both, number and length of spindle microtubules, can be fixed and are likely important for spindle function, as shown in fission yeast.
Sporozoites of P. berghei contain a fixed number of 16 subpellicular microtubules that extend from an apical polar ring at the front of the sporozoite to the nucleus at the centre of the cell. These microtubules are arranged in a typical 1 + 15 pattern and have been suggested to be important for vesicular trafficking, morphogenesis, cellular mechanics, polarity and motility of sporozoites.
All sequenced Plasmodium genomes contain one gene encoding b-tubulin and two genes encoding a-tubulins. The two a-tubulins likely arose by gene duplication from an ancestral a-tubulin. Such duplications can allow for a change in either the level or the timing of gene expression and/or to generate different functionalities arising from differences within the two coding sequences.
Expression analysis suggested that the a2-tubulin gene is expressed between one and two orders of magnitude higher than the a1- tubulin gene in blood stages. Indeed, the a2-tubulin gene appears essential and cannot be deleted, while we could readily delete the a1-tubulin gene in a wild type (WT) background and in a parasite expressing GFP and mCherry as cytoplasmic markers at different life cycle stages (see below).
Phenotype
We could readily delete the a1-tubulin gene in a wild type (WT) background and in a parasite expressing GFP and mCherry as cytoplasmic markers at different life cycle stages. Both a1-tubulin(-) parasite lines showed no defect in blood-stage growth or initial mosquito infection. However, no sporozoites were found in the midgut or salivary glands of the mosquito; thus, a main defect seems to occur at the oocyst stage. This defect could be completely rescued by complementing the gene in the a1-tubulin(-) parasite line.
Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding.
Additional information
Through analysis of a number of mutants expressing different mutated forms of alpha tubulin 1 the following was shown:
From the paper:
'Here, we quantitatively studied the impact of modulating microtubule number and length in Plasmodium, the protozoan parasite causing malaria. Using a gene deletion and replacement strategy targeting one out of two a-tubulin genes, we show that chromosome segregation proceeds in the oocysts even in the absence of microtubules. However, fewer and shorter microtubules severely impaired the formation, motility and infectivity of Plasmodium sporozoites, the forms transmitted by the mosquito, which usually contain 16 microtubules. We found that a-tubulin expression levels directly determined the number of microtubules, suggesting a high nucleation barrier as supported by a mathematical model. Infectious sporozoites were only formed in parasite lines featuring at least 10 microtubules, while parasites with 9 or fewer microtubules failed to transmit'.
- Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding.
It was shown that during late budding in the oocyst microtubules were longest and that they subsequently shrank as the sporozoites matured in the oocysts to again slightly grow as the parasites reside in the salivary gland. This suggests that, contrary to previous suggestions, cytoplasmic microtubules in Plasmodium undergo some level of shrinkage and growth, although much more slowly than in mammalian cells, on the time scale of hours to days rather than seconds.
- a1-tubulin deletion does not affect genome replication and nuclear division.
- Aberrant sporozoites are formed by intermediate numbers of microtubules.
- a2-tubulin can only partially complement a1-tubulin.
- Microtubule length and numbers affect sporozoite curvature and infectivity
Other mutants |