Engaging microtubules in successful cell division

MAP65 family proteins function as microtubule cross-linking proteins, and localise to the phragmoplast equator in the moss Physcomitrella patens (phragmoplast is a microtubule-based bipolar structure that appeared at the late stage of mitosis in plant cells). RNAi-mediated knockdown of MAP65s compromised microtubule bipolarity and affected accumulation and/or fusion of cell plate–destined vesicles at the equatorial plane.

HFSP Program Grant holders Gohta Goshima and Marcel Janson and colleagues
authored on Mon, 24 February 2014

Cytokinesis requires bipolar microtubule (MT)-based structures, the ‘central spindle’ in animals or the ‘phragmoplast’ in plants.  Given the structural similarity, the plant phragmoplast and animal central spindle are considered to be analogous structures.  In these structures, the plus ends’ MTs are directed towards the cell equator, whereas the minus ends are located near the separated sister chromosomes.  In plants, the vesicles derived from the trans-Golgi accumulate at the cell equator, possibly through motor-dependent transport along the phragmoplast MTs, which then fuse to form the cell plate.  However, it remains largely unknown how bipolarity of the phragmoplast is established and maintained and how it ensures proper cytokinesis.

Figure 1:  MAP65 is localised to the phragmoplast equator
A mitotic cell expressing Citrine (a GFP variant)-tagged MAP65 and mCherry-tubulin.  Time 0 corresponds to the timing of nuclear envelope breakdown.   

MAP65 is a conserved MT-associated protein that exhibits antiparallel MT crosslinking activity in vitro.  During cytokinesis, MAP65 is highly enriched at the phragmoplast equator in various plant cell types.  Therefore, MAP65 is the best candidate for proteins involved in crosslinking phragmoplast MTs at the equator.  However, functional analyses of the MAP65 family in Arabidopsis thaliana, which possesses 9 homologous genes in its genome, had not clarified the extent to which MAP65s contribute to phragmoplast MT crosslinking, likely because those homologous genes function redundantly.

In our paper, we analysed the function of MAP65 in the moss Physcomitrella patens, using a recently established conditional RNAi knockdown system.  We identified 5 MAP65s in P. patens, and their YFP fusions revealed that 3 of them are expressed and localised to the phragmoplast equator in a specific cell type (Figure 1).  When those 3 MAP65s were co-knocked down using an inducible RNAi system, bipolar arrangement of phragmoplast MTs was disrupted during cytokinesis, and cell plate materials failed to accumulate and/or fuse each other, resulting in cytokinesis failure.  These results indicate that MAP65 is essential for maintaining the bipolar pattern of phragmoplast MTs.  Furthermore, they demonstrated that MAP65-mediated MT interdigitation at the equator is a prerequisite for the cell plate-destined membrane trafficking.  This study also presents the usefulness of the P. patens system in determining the mitotic function of a certain gene family. 

Reference

MICROTUBULE-ASSOCIATED PROTEIN65 is essential for maintenance of phragmoplast bipolarity and formation of the cell plate in Physcomitrella patens. Kosetsu, K., de Keijzer, J., Janson, M.E., and Goshima, G. (2013). Plant Cell. 25, 4479-92.

Pubmed link