Investigating the role of the protein tyrosine phosphatase, HD-PTP, during cytokinesis

Cytokinesis is the final process of mitosis that physically divides one cell in two daughter cells and understanding this important event of the cell cycle is important for different reasons. In fact, some evidences propose a link between failure of cytokinesis and tumorigenesis; a cell that cannot correctly complete its division becomes genetically unstable and could evolve into a cancer. The first step of cytokinesis is the choice of division site. Although the mechanism that determines the position of the cleavage furrow is different between yeast and metazoans it seems that in all cases the role of microtubules is crucial . In animal cells the division plane specification happens after the segregation of chromosomes, during late mitosis. In this case microtubules specify where the cleavage furrow will be, although it's not clear whether the furrow position is determined by the overlapping astral microtubules or the anaphase spindle midzone. In the further passages, becomes important to assemble the division machinery and divide the cell; yeast and animal cells divide by the formation of an actomyosin ring, where both actin and myosin play an important role. A number of reports, however, have demonstrated that membrane trafficking is also very important both in yeast and animal cytokinesis. For example, genetic studies have proposed a vectorial protein transport in cell division, promoted by a large protein complex termed exocyst. The exocyst recognises transport vesicles and is also able to interact with the plasma membrane. In budding yeast, as in animal cells, this complex is required for cytokinesis. In this context, it is notable that there is also evidence for a role of the Endosomal Sorting Complex Required for Transport, during abscission. Normally, the ESCRT machinery is responsible for late endosome to lysosome trafficking, and one of its function is to target membrane protein for degradation. There are three ESCRT complexes; ESCRT-I and -II are important for recognizing ubiquitinylated cargo proteins, and ESCRT-III is implicated in sorting these cargo proteins and in the invagination of the endosomal membrane to form the Multi-Vesicular Body (MVB). A number of reports demonstrate that some ESCRT subunits localize to the midbody. In addition, recent studies have demonstrated a role for ESCRT-I and ESCRT III complexes in cytokinesis. Intriguingly, it has been demonstrated that Alix also has an important role during cytokinesis. This protein was known to function as an interactor of several different proteins and complexes in the MVB pathway. This protein is of a great interest because it can bind to CEP55, a protein that performs important roles in organizing the Flemming body and recruiting a series of late-acting proteins required for abscission. It has been demonstrated that both Alix and ESCRT-I are required for efficient cytokinesis. Starting from these data, we have decided to study the role of an Alix-related protein, His Domain Phosphotyrosine Phosphatase (HD-PTP). HD-PTP was known as a non-receptor protein tyrosine phosphatase, involved in driving cell migration during angiogenesis. It is a newly-discovered component of the ESCRT machinery, and has been shown to be involved in sorting cargo proteins at the endosome . In order to study the role of this protein during cytokinesis I used RNA interference to knock-down HD-PTP. Non-targeting siRNA and, in parallel, a siRNA targeting Alix were used as respectively negative, and positive control. Left protein level was checked by Western-blot and RT-PCR. Finally, cells were fixed and stained for tubulin in order to examine phenotype by Fluorescence microscopy, scoring cells for multinucleation and aberrant midbodies. Moreover, in order to perform more accurate studies and examine non-disjunction event dynamics, I used Live Imaging microscopy. For this purpose cells were synchronized in M-phase of the cell cycle using thymidine