Recent trends in plant protein complex analysis in a developmental context

Because virtually all proteins interact with other proteins, studying protein-protein interactions (PPIs) is fundamental in understanding protein function. This is especially true when studying specific developmental processes, in which proteins often make developmental stage-or tissue specific interactions. However, studying these specific PPIs in planta can be challenging. One of the most widely adopted methods to study PPIs in planta is affinity purification coupled to mass spectrometry (AP/MS). Recent developments in the field of mass spectrometry have boosted applications of AP/MS in a developmental context. This review covers two main advancements in the field of affinity purification to study plant developmental processes: increasing the developmental resolution of the harvested tissues and moving from affinity purification to affinity enrichment. Furthermore, we discuss some new affinity purification approaches that have recently emerged and could have a profound impact on the future of protein interactome analysis in plants

Differential regulation of clathrin and its adaptor proteins during membrane recruitment for endocytosis

In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2 mu or AP2 sigma partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2 sigma, but not AP2 mu, was required for SA-and tyrphostin A23-dependent inhibition of CME. Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells

Unraveling division plane positioning and endocytic protein complexes in Arabidopsis thaliana

At the onset of mitosis, plant cells reorganize their cortical microtubule array into a narrow ring encircling the nucleus until late prophase. This transient cytoskeletal structure is termed the preprophase band (PPB) and is believed to establish the plasma membrane (PM) identity at the cortical division zone (CDZ) which persists until cytokinesis. The CDZ is determined by the presence or particular exclusion of several marker proteins with a cell plate guiding function. The expanding phragmoplast mediates the directional growth of the cell plate towards the CDZ, where the cell plate anchors to the parental plasma membrane at the end of cytokinesis. Therefore, the PPB defines the division plane early in mitosis and spatially controls plant cell division. However, the molecular mechanisms by which the identity of the CDZ is established and maintained as well as the role of the division zone markers in supporting cell plate guidance remains unknown. Although a variety of proteins have thus far been described to be involved in division plane positioning, information about their biochemical properties and interconnections is largely missing. The aim of this doctoral research was to increase our current understanding of division zone demarcation in plant cells and attempt to link the known players. Because the biological function of a protein is predominantly determined by protein interaction networks, a targeted interactomics approach based on the tandem affinity purification (TAP)-technology was set up starting from a set of bait proteins recognized to play a role in division zone demarcation. The TAP technology allows a high-throughput isolation of in vivo protein complexes under native conditions and requires no prior knowledge on the protein complexes in which the bait participates. Through the co-purification of interaction partners, a division zone interactome can be built which might ultimately enable us to correlate the known players, but will definitely identify novel candidate proteins involved in the process of division plane positioning. Based on subsequent fluorescence microscopy screening, those interactors which specifically localize to division plane figures such as the preprophase band, the PM at the cortical division zone or the expanding cell plate will be selected for further functional characterization using a combination of genetic and live-cell imaging analysis. This doctoral thesis can be subdivided in two major parts, each considering a specific functional branch present in the division zone interactome. Part one (chapters 1-3) provides a general overview of the obtained division zone interactome and describes novel players and protein complexes involved in division plane positioning and division zone demarcation. This targeted interactomics approach resulted amongst others in the isolation of a TON1/TRM/FASS PP2A phosphatase (TTP) complex, which was fully characterized in a collaborative effort with the research group of Prof. Dr. David Bouchez and Dr. Martine Pastuglia (INRA, Versailles). Our data tightly connected TON1, which shows homology to human centrosomal proteins, and the PP2A B’’ regulatory subunit FASS to a heterotrimeric PP2A holo-enzyme, which is targeted to the cortical cytoskeleton by the TRM protein family Scope 4 to exert its function. Moreover, we illustrated the necessity of FASS/PP2A phosphatase activity for PPB formation. Part two of this doctoral thesis (chapters 4-6) focusses on endocytosis at the plasma membrane and how endocytosis participates in cell plate anchoring at the CDZ. In light of this question, the cell biological function of the plant-specific multi-subunit complex comprising TPLATE, isolated through the targeted division zone interactomics approach described in part one, was functionally investigated. Besides the occurrence of two distinct recruitment pathways for the TPLATE complex and CLC2 during somatic cytokinesis, a cell plate recruitment pathway and a CDZ recruitment pathway during cell plate anchoring, we show that the TPLATE complex performs a more general function in clathrin-mediated endocytosis at the PM throughout plant development. The TPLATE complex could therefore provide a functional link between plasma membrane endocytosis and the maintenance of CDZ identity

Recent Trends in Plant Protein Complex Analysis in a Developmental Context

Because virtually all proteins interact with other proteins, studying protein–protein interactions (PPIs) is fundamental in understanding protein function. This is especially true when studying specific developmental processes, in which proteins often make developmental stage- or tissue specific interactions. However, studying these specific PPIs in planta can be challenging. One of the most widely adopted methods to study PPIs in planta is affinity purification coupled to mass spectrometry (AP/MS). Recent developments in the field of mass spectrometry have boosted applications of AP/MS in a developmental context. This review covers two main advancements in the field of affinity purification to study plant developmental processes: increasing the developmental resolution of the harvested tissues and moving from affinity purification to affinity enrichment. Furthermore, we discuss some new affinity purification approaches that have recently emerged and could have a profound impact on the future of protein interactome analysis in plants

Dual localized kinesin-12 POK2 plays multiple roles during cell division and interacts with MAP65-3

Kinesins are versatile nano-machines that utilize variable non-motor domains to tune specific motor microtubule encounters. During plant cytokinesis, the kinesin-12 orthologs, PHRAGMO-PLAST ORIENTING KINESIN (POK)1 and POK2, are essential for rapid centrifugal expansion of the cytokinetic apparatus, the phragmoplast, toward a pre-selected cell plate fusion site at the cell cortex. Here, we report on the spatio-temporal localization pattern of POK2, mediated by distinct protein domains. Functional dissection of POK2 domains revealed the association of POK2 with the site of the future cell division plane and with the phragmoplast during cytokinesis. Accumulation of POK2 at the phragmoplast midzone depends on its functional POK2 motor domain and is fine-tuned by its carboxy-terminal region that also directs POK2 to the division site. Furthermore, POK2 likely stabilizes the phragmoplast midzone via interaction with the conserved microtubule-associated protein MAP65-3/PLEIADE, a well-established microtubule cross-linker. Collectively, our results suggest that dual localized POK2 plays multiple roles during plant cell division

The phragmoplast-orienting kinesin-12 class proteins translate the positional information of the preprophase band to establish the cortical division zone in Arabidopsis thaliana

The preprophase band (PPB) is a faithful but transient predictor of the division plane in somatic cell divisions. Throughout mitosis the PPBs positional information is preserved by factors that continuously mark the division plane at the cell cortex, the cortical division zone, by their distinct spatio-temporal localization patterns. However, the mechanism maintaining these identity factors at the plasma membrane after PPB disassembly remains obscure. The pair of kinesin-12 class proteins PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 are key players in division plane maintenance. Here, we show that POK1 is continuously present at the cell cortex, providing a spatial reference for the site formerly occupied by the PPB. Fluorescence recovery after photobleaching analysis combined with microtubule destabilization revealed dynamic microtubule-dependent recruitment of POK1 to the PPB during prophase, while POK1 retention at the cortical division zone in the absence of cortical microtubules appeared static. POK function is strictly required to maintain the division plane identity factor TANGLED (TAN) after PPB disassembly, although POK1 and TAN recruitment to the PPB occur independently during prophase. Together, our data suggest that POKs represent fundamental early anchoring components of the cortical division zone, translating and preserving the positional information of the PPB by maintaining downstream identity markers