The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis

Abstract

Clathrin-mediated endocytosis in plants is an essential process but the underlying mechanisms are poorly understood, not least because of the extreme intracellular turgor pressure acting against the formation of endocytic vesicles. In contrast to other models, plant endocytosis is independent of actin, indicating a mechanistically distinct solution. Here, by using biochemical and advanced microscopy approaches, we show that the plant-specific TPLATE complex acts outside of endocytic vesicles as a mediator of membrane bending. Cells with disrupted TPLATE fail to generate spherical vesicles, and in vitro biophysical assays identified protein domains with membrane bending capability. These results redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive endocytosis in plant cells.One Sentence SummaryWhile plant CME is actin independent, we identify that the evolutionarily ancient octameric TPLATE complex mediates membrane bending against high turgor pressure in plant clathrin-mediated endocytosis.

User-friendly electron microscopy protocols for the visualization of biological macromolecular complexes in three dimensions: Visualization of planta clathrin-coated vesicles at ultrastructural resolution

“…The unreliable preservation of plant CCVs in situ has hampered the characterization of these structure by EM methodologies. However, to overcome this we recently established a protoplast metal replica unroofing assay that reliably preserves large numbers of CCVs in plant cells (6, 20). Here, cells were attached to a glass coverslip, ‘unroofed’ (physically disrupted) to remove any cellular materials not associated with the plasma membrane (PM) directly attached to the coverslip, fixed, and coated with platinum; thus producing a metal replica of the inside cellular environment of plant cells which can then be examined with EM.…”

“…An elegant and extremely useful methodology to prepare biological samples suitable for EM tomography is the metal replication of unroofed cells. This allows the direct visualization of the intracellular landscape and sub-cellular organelles in vivo (2)(3)(4)(5)(6). However, to examine these samples at nanometer resolutions via transmission electron microscopy (TEM), the samples must be on an EM grid.…”

“…High throughput pseudo 3D morphology screening of CCVs SEM images of metal replicas of unroofed protoplasts, taken with a FE-SEM Merlin Compact VP equipped with an In-lens Duo detector, were obtained from Johnson et al (6). This included images of CCVs in Col-0 and WDMX2 protoplasts incubated in control (4-hour incubation at room temperature) or endocytic membrane bending disruptive (4-hour incubation at 37°C) conditions, which were collected using the same magnification (~45 k) and pixel size settings (~2.4-2.6 nm).…”

“…This included images of CCVs in Col-0 and WDMX2 protoplasts incubated in control (4-hour incubation at room temperature) or endocytic membrane bending disruptive (4-hour incubation at 37°C) conditions, which were collected using the same magnification (~45 k) and pixel size settings (~2.4-2.6 nm). These protoplasts were prepared on glass coverslips as previously described (6).…”

Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants

“…In plants, TPC is essential, as mutants of TPLATE subunits cause male sterility (Gadeyne et al, 2014; Van Damme et al, 2006; Wang et al, 2019). TPC in plants functions in clathrin-mediated endocytosis together with AP-2 and is required for clathrin-curvature, as destabilisation of the complex causes a failure for clathrin pits to transition from a flat to a curved lattice (Gadeyne et al, 2014; Johnson et al, 2021; Wang et al, 2021). Compared to amoeba, plants contain two additional TSET subunits, AtEH1/Pan1 and AtEH2/Pan1, Eps15 homology (EH) domain containing proteins homologous to human Eps15 and yeast Ede1p and Pan1p.…”