Hi Catarina.

It probably depends on which stage of mitosis you're interested in. Unfortunately, as far as I'm aware, there are no clearly defined markers for the start of mitosis let alone the various stages that make it up (hopefully, I'm wrong!). There are some markers that I've used in the (distant) past, but they will only really identify cells where mitosis is already in progress, rather than initiating.

The anti B-tubulin antibody, KMX1, identifies the mitotic spindle, which forms an elliptic shape in early anaphase and then becomes stretched and bifurcated at each end as anaphase progresses. Alternatively, you can identify cells in earlier stages of mitosis, such as prophase/metaphase, by DNA-FISH against the 177 bp minichromosome repeat, as the minichromosomes tend to congress to the centre of the nucleus at this stage (probably similar to the metaphase plate seen in other eukaryotes). Nuclear dimensions may also serve as a (very) imperfect proxy - the nucleus becomes noticeably stretched as mitosis proceeds.

Hope this is at least some help.

Sam.

Hi Sam,

Thank you for the help! I was struggling a lot to find any info on this. I asked Richard a while back and I remember him mentioning that it was indeed a part of the cell cycle that was not very well known in Tryps.

I've been calling the "mitosis" by having a stretched nucleus, but I was just looking around for markers that would complement this (and was not very lucky).

Thank you again for the help,

Catarina

I am not familiar with T. brucei, but in general you can follow mitosis by visualising DNA (DAPI stain) as it is separated during mitosis or by visualising proteins from mitotic spindle (tubulin, proteins from the chromosomal passenger complex CPC). Alternatively, you can use some mitosis- specific posttranslational modification of certain proteins (ph-Ser10 from histone H3 for example in most eukaryotic cells, but it seems to be not conserved in T.brucei).

Here are some papers which might be interesting for you:

http://www.ncbi.nlm.nih.gov/pubmed/19043568/ (describes proteins from the CPC)

http://www.ncbi.nlm.nih.gov/pubmed/24224936 (uses anti- H3K76me2 as mitotic marker)

Hope It helps you.

Thank you Eugen, I missed those ones! Best, Catarina