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Clathrin-mediated endocytosis in plants: mechanistic insight into the TPLATE REcycling compleX and its interplay with AP-2

Periodic Reporting for period 3 - T-Rex (Clathrin-mediated endocytosis in plants: mechanistic insight into the TPLATE REcycling compleX and its interplay with AP-2)

Reporting period: 2019-07-01 to 2020-12-31

The plasma membrane serves as a barrier between the cell and the outside world. It contains a whole range of proteins by which the cell can sense and communicate with the outside world.
These proteins in the plasma membrane are for example receptors (antennas) sensing the presence of pathogens or they can be transporters (doors) allowing nutrients to enter and exit the cell. Commonly, all those plasma membrane proteins are termed cargo in the endosomal trafficking pathways.
Endocytosis is the process by which the cell can control the abundance of these proteins in its plasma membrane and thereby control the cell's communication with the outside world.
This works via the specific recognition of proteins by so called adaptor complexes and the subsequent internalization of the proteins and the associated membrane into the cell's interior via vesicles which are pinched off from the plasma membrane. This process mostly works in concert with a scaffolding protein called clathrin and hence it is termed clathrin-mediated endocytosis (CME).

Whereas the process of endocytosis is quite well characterized in animal and yeast cells, mechanistically, this process remains poorly understood in plants. Moreover, recent evidence from my group revealed that plant cells, in contrast to other Eukaryotes, have retained an evolutionary ancient adaptor complex controlling this process, the TPLATE complex (TPC).
Whereas yeast and animal cells have evolutionary acquired alternative solutions avoiding the need of this ancient adaptor complex, plants for some reason did not. Answering the question why plants specifically retained this complex throughout Eukaryotic evolution, is one of the main biological questions we are pursuing in my group.

Insight into how endocytosis operates in plants is an essential step toward being able to modulate hormone and defense signaling, nutrient uptake, toxin avoidance and cell wall formation.
The aim of the T-REX project is to mechanistically understand clathrin-mediated endocytosis in plants, which will also provide insight into evolutionary aspects of membrane trafficking mechanisms. The project combines high-end proteomic approaches with live-cell imaging and structural biology. It provides a good balance between (1) challenging, high-end and therefore more risky, experiments which will require optimization and (2) more straightforward experiments allowing to clearly address testable hypotheses.

More specifically, this project aims to tackle plant endocytosis by answering the following biological questions:

- Is the evolutionary retention of the TPC in plants causal to specific cargo recognition?
- What are the spatio-temporal dynamics of 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 with functionality?
The work performed is listed in detail in the summary of the major achievements. As I consider the unpublished work performed and the obtained results as largely confidential, I prefer not to repeat them here.
The plant plasma membrane (PM) contains a wide range of receptors, channels and other membrane proteins that control nutrient uptake and mediate communication of the cell with the outside world. These PM proteins perceive for example hormonal stimuli, nutrient availability or pathogen attack and transfer these signals over the PM to the plant cell via signaling cascades that ultimately control plant growth and development.
Modulation of signaling pathways starting from the PM requires control over the PM proteome. While anterograde secretory pathways (exocytosis) deposit PM proteins, their removal depends on retrograde transport by endocytosis, in which PM material and extracellular ligands, together termed cargo, are predominantly internalized using coated vesicles. This mechanism provides the cell with an immediate and non-transcriptional way to react to various stimuli. Clathrin-mediated endocytosis (CME), defined by the involvement of the scaffold protein clathrin guiding the formation of a cage around the invaginating membrane, is the best characterized endocytic pathway in eukaryotes.
In contrast to other eukaryotes, knowledge of cargo recognition and the precise regulation of CME in plants is only beginning to emerge and there are still many gaps in our understanding of this process.
Although several core components of CME such as the Adaptor Protein Complex 2 (AP-2), dynamin-like proteins and clathrins are conserved across the eukaryotic Kingdoms, a broad range of effectors are absent from plants. On the other hand, plant-specific effectors of CME also exist, strongly suggesting the evolutionary divergence of a plant-specific CME mechanism.

The Adaptor Protein 2 complex (AP-2), the core adaptor complex in animal CME, was recently demonstrated to also be involved in plant CME. However, while this complex is essential in animals, loss of AP-2 subunits in plants only results in mildly aberrant phenotypes. Recently, I reported the discovery of a novel, eight-core-subunit complex (the TPLATE complex, TPC) that acts as the key plant endocytic adaptor complex. Similar to AP-2, all TPC subunits are recruited to dynamic foci at the PM. However, in contrast to AP-2, knock-outs in all tested subunits of the TPC are lethal. In line with a major role for the TPC in plant CME, induced silencing of TPC subunits impaired internalization of well-known endocytic cargo proteins as well as endocytic tracers.
Live-cell imaging revealed three populations of endocytic foci at the PM: foci which recruited only the TPC or AP-2 during their life-time and foci which recruited both. The number of foci recruiting only the TPC outnumber the amount of AP-2-only foci at the PM. These observations suggest the existence of at least two independent mechanisms for cargo recognition in plants. These might reflect differential cargo and/or differential states of common cargo (e.g. representing constitutive endocytosis versus ligand-induced or recycling versus degradation).
The core TPC consists out of eight proteins for which no obvious homologs can be identified in animal or yeast genomes although an extensive structural homology-based search recently did discover a similar complex (TSET) in the slime mold Dictyostelium. The TPC/TSET complex represents an ancient adaptor complex which is lost completely in the lineage leading to animal and fungal cells (opisthokonts). Although still present in Dictyostelium, it appears no longer functional or essential there either. Moreover, Hirst and co-workers put forward the hypothesis that the opisthokont muniscin proteins which act as an endocytic hub in CME and activate AP-2 are the sole remnants derived from this ancient complex. Although strongly diverged, the TPC subunit TML is the closest plant relative to the opisthokont muniscin family and also contains a C-terminal μ Homology Domain (μHD) involved in membrane interactions, cargo recognition and recruitment of accessory proteins.
The fact that plants are likely the only Kingdom where this evolutionary ancient endocytic complex remained essential demonstrates fundamental differences between plant and opisthokont endocytic mechanisms. Detailed analysis of TPC ultrastructure, assembly, cargo-recognition, and its relationship with AP-2 will yield important insights into evolutionary aspects of CME in eukaryotes.
This project will resolve long-standing problems in the understanding of how plant CME operates via a multidisciplinary approach. Firstly, I will use a genome-wide proteomics-based approach to evaluate cargo-specificity of both the TPC and AP-2 adaptor complexes. This will explain the differential mutant phenotypes of both complexes. The expected results at the end of the project are to be able to attribute the recognition and internalization of specific cargo proteins (or their destiny) to one of both adaptor complexes. The nature of these cargoes should allow to explain the differences in mutant phenotypes observed between AP-2 and TPC. Moreover, the proteomics approach will not only identify cargoes, but very likely also several unknown interactors/modulators of these adaptor complexes. The outcome of this part of the project therefore will also be an expansion of the interaction network surrounding the both adaptor complexes operating at the PM and a substantial increase in knowledge on how CME in plant operates and the players involved.
Secondly, I will use dynamic live-cell imaging, interaction analyses and structural biology to dissect adaptor complex assembly and to determine the ultrastructure of the entire TPC as well as individual subunits/domains, yielding functional insight. The expected result of this part of the project will be to have a validated structural model of the TPLATE complex. Depending on the success of certain approaches, this model will have high or low overall resolution, next to high resolution of specific domains which are independently resolved via recombinant protein domain purification approaches followed by X-ray or NMR-based structure determination. Nevertheless, it will likely be the first structural model of a plant adaptor complex and it will provide us with a view on endocytosis throughout eukaryotic evolution. This model will serve a starting point to understand cargo recognition at the molecular level, which in turn is essential to be able to modulate agriculturally important processes such as nutrient and water uptake, biomass production and pathogen defense. Moreover, structural insight into a complex which appears only essential in plants will also allow to design specific screening strategies to identify chemicals which specifically disrupt this complex’ function and this thus represents a completely novel mode of action for novel herbicide development.