Towards mechanistic insight into the TPLATE adaptor complex in plant endocytosis

icoontje TPLATE complexEndocytosis controls cellular perception of the outside world but how this is regulated in plants remains largely unknown. Moreover, recent evidence shows that plants, in contrast to other Kingdoms, have retained an evolutionary ancient mechanism that controls this process. Gaining mechanistic insight into endocytosis in plants is an essential step towards being able to modulate plant signaling pathways and towards understanding the evolution of eukaryotic membrane trafficking mechanisms.

TPLATE has been identified as a subunit of an octameric protein complex (The TPLATE complex, TPC) driving clathrin-mediated endocytosis at the plasma membrane in plant cells, working likely independently as well as in concert with the evolutionary conserved AP-2 heterotetrameric adaptor protein complex (Gadeyne et al., 2014). This complex represents an evolutionary ancient adaptor module which, although still present in Dictyostelium but not functional anymore, has been completely lost in the lineage leading to animal and yeast cells (Hirst et al., 2014). In contrast to other effectors of endocytosis like clathrin, all TPC subunits localize exclusively to the plasma membrane and accumulate to highly dynamic endocytic foci which show no lateral movement, allowing to accurately measure the dwell-time of their recruitment via kymograph analysis.

 

Time lapse movie of TPLATE-GFP in the tplate (-/-) background in Arabidopsis hypocotyl cells.

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TPLATE-GFP

(top) Co-localization of TPLATE-GFP and CLC2-TagRFP in Arabidopsis root epidermal cells showing specific recruitment of TPLATE to the PM and (bottom) kymograph representation allowing to measure the dwell-time of TPLATE-containing endocytic foci.

The overall objective of the current research in the group is to gain a deep mechanistic insight into the developmentally essential process of CME in plants. This will enable us to specifically modulate the abundance of plasma membrane proteins involved in nutrient uptake, toxin avoidance, cell wall formation but also hormone and defence responses. Understanding TPC-dependent CME will also provide insight into evolutionary aspects of endocytosis. In the framework of an ongoing ERC project, we are addressing the following questions with respect to unravel the mechanism of TPC-dependent endocytosis in plants:

  • Is the evolutionary retention of the TPC in plants causal to specific cargo recognition?
  • What are the spatio-temporal dynamics of TPC and CME effectors at the plasma membrane?
  • How does acute removal of TPC subunits affect complex recruitment and CME?
  • How is the TPC organized at the structural level?
  • Which interactions occur and can we couple subunit/domain structures to functionality?

To answer these questions, we combine state-of-the art proteomics with highly dynamic multi-color live cell imaging and structural biology, including protein crystallization and single particle structural EM analysis.

As endocytosis of transmembrane proteins (cargo) is initiated by the recognition of the cargo by adaptor proteins, elucidating the mechanisms that determine the interaction between the plant-specific TPC, the AP-2 complex and their cargo will allow modulation of the internalization of certain cargo proteins, including those involved in division plane determination and identity maintenance. Identification of TPC and AP-2 complex cargo proteins will be performed using targeted biotinylation (bioID, Roux et al., 2012) where a promiscuous biotin ligase will cause biotinylation of interactors of both adaptor complexes. Biotinylated proteins will subsequently be purified and identified using mass spectrometry.

To visualize the spatio-temporal dynamics, we are generating fluorescently tagged functional fusions of TPC subunits and we are combining this with approaches to inhibit the TPC function (inducible silencing, over expression of DN constructs). To unravel the structural organization of the TPC, we are modeling the TPC together with identifying conserved domains of which we will address the function by performing mutant complementation experiments. We are establishing assays to purify the TPC from plant cells or heterologous systems like Pichia pastoris or sf9 insect cells to be able to determine its ultrastructure via single particle EM analysis (negative stain and cryo-EM). Finally, we are looking at interactions among the TPC subunits by Y3H as well as co-IP experiments combined with chemical genetics approaches to disrupt interactions among TPC subunits.

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.
  • Hirst J, Schlacht A, Norcott JP, Traynor D, Bloomfield G, Antrobus R, Kay RR, Dacks JB, Robinson MS. (2014). Characterization of TSET, an ancient and widespread membrane trafficking complex. Elife. 3:e02866.
  • Roux KJ, Kim DI, Raida M, Burke B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 196: 801-810.