Introduction

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The discovery of fluorescent proteins allowed monitoring intracellular processes in real time and brought dynamic behavior and transitions as novel parameters to be measured by microscopic imaging. The advanced live cell imaging group is cornered on the use of various tools to achieve dynamic imaging of plant intracellular processes. As model systems, we use tobacco Bright Yellow-2 culture cells, Arabidopsis seedlings, Nicotiana benthamiana epidermal leaf cells and we also work with the moss Physcomitrella patens. Initially, the focus lay on imaging the events associated with cellular division such as preprophase band formation and positioning, division zone establishment and cell plate formation. We continue on this topic by elucidating the role of the plant aurora kinases as we have shown that they function in division plane determination during formative divisions (Van Damme et al., 2011a).

Currently, the major research topic in the group aims to elucidate the highly dynamic process of endocytosis at the plasma membrane in plant cells where several players are recruited in a highly specific order. Following the recruitment of early adaptor proteins which recognize transmembrane cargo proteins such as receptors and channels destined for internalization, endocytosis progresses through the formation and fission of a coated pit, allowing the physical internalization of these membrane proteins together with ligands and lipids.

Our cell biological and structural approaches aim to identify and elucidate the recruitment of the endocytic players at the PM, largely focusing on the TPLATE adaptor complex (Gadeyne et al., 2014), to map interactions among the players and to investigate how cargo is being recognized.

A long-term aim of the Advanced Live Cell Imaging Group is to be able to modify plant architecture by controlling the orientation of the plane of cell division in both proliferative and formative divisions. To do so requires an elucidation and profound understanding of the mechanisms at play which orient and subsequently maintain the division plane. We are combining state-of-the-art cell biological, genetic, structural and proteomic approaches to achieve this goal.

Due to the presence of a cell wall which surrounds and glues plant cells together, plants rely on the interplay between oriented cell divisions and elongation to establish their overall body plan. Next to proliferative (symmetric) divisions, plants utilize formative (asymmetric) divisions to establish new organs. In the majority of higher plant cells, both proliferative and formative division planes are determined by the formation of a plant-specific cytoskeletal structure termed the preprophase band (PPB). This structure encircles the premitotic nucleus and determines the future division plane long before the cell actually divides. The PPB is however only a transient structure as it disappears prior to nuclear envelope breakdown and its exact function in establishing the future division plane remains still not fully understood.

MAP65-5-GFP PPB projection C001A001

3D Projection of a tobacco Bright Yellow-2 (BY-2) cell expressing MAP65-5-GFP, which marks the dense preprophase band encircling the premitotic nucleus.

The PPB sets the stage for division plane identity establishment via the formation of the cortical division zone (CDZ). The CDZ is a specific region in the plasma membrane (PM) at the position of the PPB with a different composition and serves to remember the position where the cell should divide following PPB degradation.

How the identity of this CDZ is established and maintained until the end of cytokinesis remains unknown, but this involves the exclusion of certain negative markers such as actin and KCA1 (Van Damme, 2009), the specific recruitment of positive early CDZ markers like Tangled, POK1 and RanGAP1 (Rasmussen et al., 2011; Lipka et al., 2014), and late CDZ markers like TPLATE (Van Damme et al., 2006; 2011b). Moreover, a localized increase in endocytic activity (Karahara et al., 2009) is associated with the CDZ underlying the PPB and several endocytic markers indeed specifically remain accumulated there following PPB degradation.

Confocal sections of tobacco Bright Yellow-2

Confocal sections of tobacco Bright Yellow-2 (BY-2) cells at metaphase showing CDZ accumulation of the positive marker POK1 (green, MTs are red; A) as well as effectors of endocytosis (B).

Generating knowledge on how endocytosis functions in plants will provide a framework for the development of novel tools to interfere with endocytosis in plants. Application of these tools in dividing cells will allow us to dissect the function of endocytosis in establishing and maintaining the cortical division zone PM domain required for division plane determination and will allow us to modulate this process to alter plant architecture.

References

  • Gadeyne A, Sánchez-Rodríguez C, Vanneste S, Di Rubbo S, Zauber H, Vanneste K, Van Leene J, De Winne N, Eeckhout D, Persiau G, Van De Slijke E, Cannoot B, Vercruysse L, Mayers JR, Adamowski M, Kania U, Ehrlich M, Schweighofer A, Ketelaar T, Maere S, Bednarek SY, Friml J, Gevaert K, Witters E, Russinova E, Persson S, De Jaeger G, Van Damme D. (2014). The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants. Cell. 156, 691-704.
  • Karahara I., Suda J., Tahara H., Yokota E., Shimmen T., Misaki K., Yonemura S., Staehelin L.A. and Mineyuki Y. (2009). The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis. Plant J. 57, 819-831.
  • Lipka E, Gadeyne A, Stöckle D, Zimmermann S, De Jaeger G, Ehrhardt DW, Kirik V, Van Damme D, Müller S. (2014) The Phragmoplast-Orienting Kinesin-12 Class Proteins Translate the Positional Information of the Preprophase Band to Establish the Cortical Division Zone in Arabidopsis thaliana. Plant Cell. 26, 2617-2632.
  • Rasmussen C.G., Humphries J.A. and Smith L.G. (2011). Determination of symmetric and asymmetric division planes in plant cells. Annu. Rev. Plant Biol. 62, 387-409.
  • Van Damme D., Coutuer S., De Rycke R., Bouget F.Y., Inzé D. and Geelen D. (2006). Somatic cytokinesis and pollen maturation in Arabidopsis depend on TPLATE, which has domains similar to coat proteins. Plant Cell 18, 3502-3518.
  • Van Damme D. (2009). Division plane determination during plant somatic cytokinesis. Curr. Opin. Plant Biol. 12, 745-751.
  • Van Damme D., De Rybel B., Gudesblat G., Demidov D., Grunewald W., De Smet I., Houben A., Beeckman T. and Russinova E. (2011a). Arabidopsis alpha Aurora kinases function in formative cell division plane orientation. Plant Cell 23, 4013-4024.
  • Van Damme D., Gadeyne A., Vanstraelen M., Inzé D., Van Montagu M.C., De Jaeger G., Russinova E. and Geelen D. (2011b). Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways. Proc. Natl. Acad. Sci. USA 108, 615-620.