Cytoskeleton-associated protein complexes during plant cell division

In recent years it has become apparent that major cell biological processes are not dependent on individual proteins, but are carried out by assemblies of protein molecules. Different proteins work together in a highly coordinated way, resembling “molecular machines”. Several of these molecular machines can be found during division, when protein complexes precisely orchestrate profound changes in the structure and physiology of the cell. The plant microtubule (MT) cytoskeleton plays an essential role in cell division and undergoes fast rearrangements from cortical microtubules to a preprophase band, spindle and phragmoplast. This process requires the cooperation of several MT-associated proteins (MAPs). Although the knowledge on plant MAPs is gradually increasing, not much is known yet about the interactions between these MAPs, nor about the processes that regulate their activity, such as phosphorylation. In animal cells it is already described that the mitotic Aurora kinase is an important player in the cytoskeletal organization during division. Aurora kinase is therefore often compared to a conductor of a symphonic orchestra, interacting with several microtubule-binding partners, and coordinating the transitions through the different phases of the mitotic symphony. In plants however, not much is known yet about the function of the Aurora kinases or their interacting proteins. Consequently, in this research we aimed to identify MAP protein complexes that function together in the successful execution of mitosis and cytokinesis, specifically focusing on AURORA1 complexes. Yeast two-hybrid library screens and Tandem Affinity Purification experiments were performed to identify interaction partners (Chapter 2). To narrow down the resulting set of candidate interacting proteins, their GFP-localization was followed in dividing BY-2 cells. This strategy resulted in identification of candidate proteins that associate with the cytoskeleton or cell plate during cell division. In Chapter 3, the interaction between AURORA1 (AUR1) and its novel interacting partner, ARCTICA1 (ARC1) is analyzed in more detail. We provide evidence that ARC1 is an in vitro substrate of AUR1. Besides localizing to kinetochores and the cell plate during cell division, ARC1 associated with the plasma membrane in a polar manner. This membrane association was further characterized in Chapter 4. Finally, a similar strategy was followed to study the binding partners of the EB1 (End Binding 1) protein, that is known to form an interaction hub at the microtubule plus end in human cells (Chapter 5). Our interaction assays identified that the EB1 family of microtubule plus-end binding proteins dimerize in plants, and we further investigated the function of EB1 dimerization in the EB1 plus-end complex assembly

Nuclear-localized subtype of end-binding 1 protein regulates spindle organization in Arabidopsis

End-binding 1 (EB1) proteins are evolutionarily conserved plus-end-tracking proteins that localize to growing microtubule plus ends where they regulate microtubule dynamics and interactions with intracellular targets. Animal EB1 proteins have acidic C-terminal tails that might induce an autoinhibitory conformation. Although EB1 proteins with the same structural features occur in plants (EB1a and EB1b in Arabidopsis thaliana), a variant form (EB1c) is present that lacks the characteristic tail. We show that in Arabidopsis the tail region of EB1b, but not of EB1c, inhibits microtubule assembly in vitro. EB1a and EB1b form heterodimers with each other, but not with EB1c. Furthermore, the EB1 genes are expressed in various cell types of Arabidopsis, but the expression of EB1c is particularly strong in the meristematic cells where it is targeted to the nucleus by a nuclear localization signal in the C-terminal tail. Reduced expression of EB1c compromised the alignment of spindle and phragmoplast microtubules and caused frequent lagging of separating chromosomes at anaphase. Roots of the eb1c mutant were hypersensitive to a microtubule-disrupting drug and complete rescue of the mutant phenotype required the tail region of EB1c. These results suggest that a plant-specific EB1 subtype has evolved to function preferentially on the spindle microtubules by accumulating in the prophase nucleus

Sequence-specific protein aggregation generates defined protein knockdowns in plants

Protein aggregation is determined by short (5-15 amino acids) aggregation-prone regions (APRs) of the polypeptide sequence that self-associate in a specific manner to form beta-structured inclusions. Here, we demonstrate that the sequence specificity of APRs can be exploited to selectively knock down proteins with different localization and function in plants. Synthetic aggregation-prone peptides derived from the APRs of either the negative regulators of the brassinosteroid (BR) signaling, the glycogen synthase kinase 3/Arabidopsis SHAGGY-like kinases (GSK3/ASKs), or the starch-degrading enzyme alpha-glucan water dikinase were designed. Stable expression of the APRs in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) induced aggregation of the target proteins, giving rise to plants displaying constitutive BR responses and increased starch content, respectively. Overall, we show that the sequence specificity of APRs can be harnessed to generate aggregation-associated phenotypes in a targeted manner in different subcellular compartments. This study points toward the potential application of induced targeted aggregation as a useful tool to knock down protein functions in plants and, especially, to generate beneficial traits in crops

Somatic cytokinesis and pollen maturation in Arabidopsis depend on TPLATE, which has domains similar to coat proteins

TPLATE was previously identified as a potential cytokinesis protein targeted to the cell plate. Disruption of TPLATE in Arabidopsis thaliana leads to the production of shriveled pollen unable to germinate. Vesicular compartmentalization of the mature pollen is dramatically altered, and large callose deposits accumulate near the intine cell wall layer. Green fluorescent protein (GFP)-tagged TPLATE expression under the control of the pollen promoter Lat52 complements the phenotype. Downregulation of TPLATE in Arabidopsis seedlings and tobacco ( Nicotiana tabacum) BY-2 suspension cells results in crooked cell walls and cell plates that fail to insert into the mother wall. Besides accumulating at the cell plate, GFP-fused TPLATE is temporally targeted to a narrow zone at the cell cortex where the cell plate connects to the mother wall. TPLATE-GFP also localizes to subcellular structures that accumulate at the pollen tube exit site in germinating pollen. Ectopic callose depositions observed in mutant pollen also occur in RNA interference plants, suggesting that TPLATE is implicated in cell wall modification. TPLATE contains domains similar to adaptin and b-COP coat proteins. These data suggest that TPLATE functions in vesicle-trafficking events required for site-specific cell wall modifications during pollen germination and for anchoring of the cell plate to the mother wall at the correct cortical position

Chemical inhibition of a subset of Arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling

Glycogen synthase kinase 3 (GSK3) is a key regulator in signaling pathways in both animals and plants. Three Arabidopsis thaliana GSK3s are shown to be related to brassinosteroid (BR) signaling. In a phenotype-based compound screen we identified bikinin, a small molecule that activates BR signaling downstream of the BR receptor. Bikinin directly binds the GSK3 BIN2 and acts as an ATP competitor. Furthermore, bikinin inhibits the activity of six other Arabidopsis GSK3s. Genome-wide transcript analyses demonstrate that simultaneous inhibition of seven GSK3s is sufficient to activate BR responses. Our data suggest that GSK3 inhibition is the sole activation mode of 1311 signaling and argues against GSK3-independent BR responses in Arabidopsis. The opportunity to generate multiple and conditional knockouts in key regulators in the BR signaling pathway by bikinin represents a useful tool to further unravel regulatory mechanisms