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Dual Localisation Kinesin

Here, you find our publication on this mysterious motor: Xu et al. 2018.

A short explanation for the general public can be found here.

Evolution of the cytoskeleton

The plant cytoskeleton differs considerably from that of other eukaryotes - for instance, there are no intermediate filaments, no nuclear lamina, no integrins that link the cytoskeleton with the extracellular matrix (although there are analogous structures). Most importantly, the centrioles that are indispensable for animal mitosis, are not needed for cell division in plants - the spindle is organised at more amorphous microtubule-organising centers (MTOCs) near the nuclear lamina. Nevertheless, algae still have a centriole, to drive flagellae, and lower land plants (mosses and ferns) still have them as well, but only in the spermatozoid. The invention of pollen tubes render the spermatozoid dispensible. Probably for this reason, seed plants have lost dynein motors. How can they carry on with minus-end directed movements then? At the same time, a novel class of kinesins, the KCH kinesins, explodes in variability. Are the KCHs the evolutionary descendants of dyneins in plants?

A second interesting route are division proteins of prokaryotes, the so called FtsZ, that seem to be the evolutionary ancestors of the tubulins. Plants still harbour FtsZ homologues, whereas animals have lost them completely. The reason is linked with organelle division. The FtsZ proteins in prokaryotes form a division ring required to separate the daughter cells. Plastids have derived from cyanobacteria and still use FtsZ for plastid division (mitochondria had been "domesticated" much earlier and have lost FtsZ). The gene for FtsZ is not encoded in the plastid genome, however, but was transferred into the nucleus, which means that the protein has to be imported into the plastid (by a specific plastid signature).

Your task is to investigate by means of database searching and basic bioinformatic tools one evolutionary aspect of cytoskeletal evolution in plants: While textbooks tell that the cytoskeleton is absent from the interphase nucleus, there is increasing evidence that both actin and tubulin, along with some of their associated proteins, can sometimes be found in the nucleus. What are they doing there? Just an accident or linked with a real function? We have recently found a novel class-XIV kinesin that can move into the nucleus in response to cold stress (more...). This protein can bind specific motifs on the DNA and overexpression of this protein specifically alters the expression of cold box factor 4, a key regulator for cold adaptation. We therefore think that this kinesin, which we baptised Dual Localisation Kinesin, is part of a thermometer system. To understand this protein more deeply, we would like to know a bit about its evolutionary history. Likewise, we would like to know, whether it exists also in other Angiosperms of interest, such as grapevine, cereals other than rice, but also in gymnosperm, ferns, mosses, or algae. This can be done by constructing a phylogenetic tree. This is your task.

How to do it? You have first to get your raw material - catch many (!) protein sequences from different organisms of different levels of evolution: cyanobacteria, algae, mosses, ferns, gymnosperms. angiosperms. Then you have to do an alignment (CLUSTAL W), either web-based our after downloading appropriate software to your computer. From this you can then do a Neighbor-Joining (NJ) tree, which helps you to see similarities.

There are many ways to enter the world of protein evolution. One of the most appropriate is the Swissprot database, which is focussing on proteins (i.e. you work only with protein sequences, which has the advantage that you do not need to care for introns, promotors or non-coding sequences). Invest some time to get familiar with this web-based tool, it is useful for your future as biologists:

Here the entrance to the Uniprot database of Swissprot

you can search either by typing into the mask the name of the protein along with the name of the plant (e.g. FtsZ Arabidopsis). In your case, you can get the accession number from the Xu et al. 2018 paper. You will then get access to hits and you will find here not only the sequence (in FASTA Format), but also information on domains found (InterPRO or Pfam) in the protein, Information on functions, mutants and crystall structure (if available). Don't get lost! There is also a button, where you can blast to find homologues. Get a couple of the different organismic groups (it might be also a good idea to decrease the stringency of the blast search to see also more remote homologues). There is no point to get 55 Angiosperm sequences and none of the other taxonomic groups, try to collect around 10 for each systematic group (in total around 50 that is sufficient).

For alignment, there are web-based ClustalW tools (for instance this one run by the EMBL-EBI called ClustalW2-Phylogeny), where you also can have the tree plotted.

If you want to know more clearly, what you are doing, you can also use Freeware to do it, for instance the MEGA Software that has many Options for phylogenetic analysis of both protein and DNA sequences and is quite intuitive in its use.