Year: 2014
Type: dissertation
Abstract: Cytokinesis is the partitioning of the cytoplasm following nuclear division.In plants, cytokinesis starts at the middle of a dividing cell with the formation of a specialized membrane compartment, the cell plate.Tethering factors, which mediate the first specific contact between donor and acceptor membranes, are considered to play a significant role in cell plate formation.Although many players involved in cytokinesis have been identified over the past few decades, little was known about the role of tethering factors in plant cell division.Moreover, cell plate biogenesis and identity was poorly understood.In this study, a reverse genetic approach was used to identify tethering factors required for cytokinesis in Arabidopsis thaliana.The major focus was on the TRAPPI and TRAPPII tethering complexes, which are thought to mediate flow through the Golgi and at the trans-Golgi network, as well as on the GARP tethering complex, which is thought to tether endocytotic vesicles to the trans-Golgi network.Weak cytokinesis defects were found in TRAPPI mutants and strong cytokinesis defects in TRAPPII mutants.Electron microscopy on TRAPPII mutant seedlings revealed that dividing cells in these mutants are often multinucleate, with gapped or incomplete cross walls.In addition, vesicles accumulating at the equator of dividing cells failed to assemble into a cell plate.Additionally, phenotypic analysis of TRAPPII mutants showed that the TRAPPII complex is implicated in endocytosis.No evidence for cytokinesis defects was found in GARP insertion lines.Live imaging showed that the TRAPPII complex localizes to the cell plate throughout cytokinesis.A comparison of the localization dynamics of TRAPPII complex and of another tethering complex, the exocyst, in dividing cells was performed.This showed that these two tethering complexes have a sequential appearance, with brief overlap at the onset and end of cytokinesis.Moreover, immunoprecipitation and mass spectrometry provided an evidence that these two complexes interact physically in dividing cells.Immunostain analysis showed that the TRAPPII is required for cell plate assembly and exocyst for cross wall maturation.Additionally, we demonstrated that the TRAPPII complex is required for protein sorting at the cell plate.Summary 3 Taken together, we show that TRAPPII plays an important and significant role in plant cytokinesis.We propose that the coordinated action of the TRAPPII and exocyst complexes might orchestrate not only plant but also animal cytokinesis.V Voltage VAMP Vesicle associated membrane proteins VHA-a1 Vacuolar H ATPase subunit a1 Vps Vacuolar sorting wt Wild type Y2H Yeast two hybrid YFP Yellow fluorescent protein TRAPPII tethering complexTRAPP stands for transport protein particle.The multi-subunit TRAPP complex was first identified in yeast, in a genetic screen where the TRAPP blocked early in transport 3 (Bet3p) subunit was found to interact with the ER to Golgi t-SNARE Bet1p.Biochemical approaches using tagged Bet1p led to TRAPP complex isolation (Sacher et al., 1998).Later, gel filtration chromatography of yeast lysates resulted in identification of two forms of TRAPP tethering complex: the TRAPPI and the TRAPPII.Each of them acts at different stage in membrane trafficking (Sacher et al., 2001).Both complexes consist of six-core subunits: Bet3 (two copies), Bet5, TRAPP subunit 20
Author: Katarzyna Rybak
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
Funder: Deutsche Forschungsgemeinschaft
OpenAlex ID: W2743734785