# Clathrin assemblies at a glance

Stéphane Vassilopoulos, Guillaume Montagnac

► To cite this version:

Stéphane Vassilopoulos, Guillaume Montagnac. Clathrin assemblies at a glance. Journal of Cell Science, 2024, 137 (8), 10.1242/jcs.261674. hal-04677886

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assembled *in vitro* was obtained using electron cryomicroscopy, as shown by PDB IDs 3IYV and 1XI4 (Fotin et al., 2004). Clathrin triskelia are recruited by clathrin adaptors that trigger clathrin-coated vesicle budding (Keen, 1990; Kirchhausen, 1999; Pearse and Robinson, 1990). Because of their shape and composition, these membrane invaginations are termed clathrin-coated pits (CCPs). Decades of research have highlighted the fine-tuned mechanisms orchestrating CCP assembly, dynamics, maturation and function (Briant et al., 2020; Robinson, 2015). However, clathrin assemblies are not restricted to CCPs at the PM. Clathrin recruitment is controlled by a plethora of adaptors and accessory proteins that determine the specific functions and dynamics of these assemblies not only at the PM but also on internal membranes, such as the Golgi, endosomes or even on membrane-less compartments, such as the kinetochore k-fibers during mitosis (Royle, 2011; Royle et al., 2005). The most well-studied function of clathrin assemblies is to regulate the intracellular trafficking of different cargoes. Over the years it has become more and more clear that clathrin lattices can assemble into a variety of structures at different subcellular locations and can adopt different ultrastructural organizations, thus fulfilling functions that extend beyond endocytosis. In this Cell Science at a Glance article and the accompanying poster, we review the different types of clathrin assemblies and highlight the diversity of their functions. This article focuses on clathrin structures involving membranes and for detailed information on clathrin assembly at membrane-less compartments, such as the kinetochore k-fibers during mitosis, we refer readers to excellent previous reviews (Royle, 2011, 2012; see also Ryan et al., 2021). We will start by discussing the canonical functions of clathrin in regulating intracellular transport from the PM and intracellular compartments, and continue with its roles in unconventional clathrin-coated structures (CCSs), such as flat clathrin plaques and tubular clathrin-adaptor protein 2 (AP-2) lattices, as well as bilayered endosomal coats consisting of clathrin and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; also known as HGS).

## Clathrin-coated pits on the PM, the TGN and recycling endosomes

Clathrin-coated pits and vesicles were discovered using EM by Roth and Porter in their studies on yolk protein uptake in mosquito oocytes (Roth and Porter, 1964). They observed 'bristle-coated' pits of ~20 nm in diameter pinching off from the cell membrane, which then became vesicles that carried the adsorbed yolk protein into the oocyte. Subsequent pioneering work from John Heuser's laboratory combined deep-etching with rotary shadowing and EM to capture the formation of CCPs in their close-to-native state in numerous cell types and tissues (Heuser, 1980, 1989; Heuser and Kirchhausen, 1985). Although our knowledge of the roles of clathrin has greatly increased in the past 60 years, the canonical function of clathrin-coated membrane structures is still understood to be to facilitate intracellular receptor trafficking. In the case of endocytosis from the cell surface, clathrin is recruited at the PM by the heterotetrameric AP-2 complex (comprising AP2A1 or AP2A2, AP2B1, AP2M1 and AP2S1) (Robinson and Pearse, 1986). Along with numerous additional partners, clathrin and AP-2 assemble into CCPs with sizes varying from 60 to 120 nm in diameter that progressively bend the PM to generate receptor-containing clathrin-coated vesicles (see poster) (Heuser and Kirchhausen, 1985; Moulay et al., 2020; Sochacki et al., 2021).

However, CCPs do not form only on the cytoplasmic side of the PM. Using thin-section EM to observe the absorption of horseradish

peroxidase in the epithelium of the rat vas deferens, clathrin has also been found at the trans-Golgi network (TGN), where it assembles into 80 nm-wide bristle-coated vesicles to transport hydrolytic enzymes from the Golgi complex to multivesicular bodies (Friend and Farquhar, 1967). It was later shown that clathrin recruitment at the TGN is controlled by the heterotetrameric AP-1 clathrin adaptor complex (comprising AP1B1, AP1G1 or AP1G2, AP1M1 or AP1M2, and AP1S1, AP1S2 or AP1S2) (see poster; Ahle et al., 1988; Pearse and Robinson, 1990) and that the GTPase Arf1 activates cargo binding by unlocking an open conformation of AP-1. Arf1 binds to multiple binding sites on AP-1 dimers to allosterically drive both its recruitment to the TGN and binding of cargo (Ren et al., 2013).

CCPs can also bud from endosomal recycling tubules to regulate the transport of some receptors back to the PM (see poster) (Futter et al., 1998; Stoorvogel et al., 1996). Interestingly, structures termed 'gyrating-clathrin' (G-clathrin) (Zhao and Keen, 2008), which are enriched in AP-1 and Golgi-localized, γ-ear-containing, Arf-binding protein (GGA1), have been characterized on endosomes and are involved in β1 integrin and transferrin receptor recycling (Majeed et al., 2014). Thus, specific adaptors dictate the recruitment of clathrin onto these different types of membranes. These adaptors provide specificity towards different membranes by binding to phosphoinositides that are only found at their target membranes (see poster). Phosphatidylinositol 4,5-bisphosphate (PIP2) is found exclusively at the PM and is required for CCP assembly (Jost et al., 1998), whereas phosphatidylinositol 3-phosphate (PI3P) is required for Hrs-dependent clathrin assembly into bilayered structures at the endosomal membrane (Wenzel et al., 2018) and phosphatidylinositol 4-phosphate (PI4P) recruits AP-1 and clathrin at the TGN (Wang et al., 2003). In addition to these canonical functions that regulate budding, clathrin has also been shown to form unconventional flat lattices and tubular structures at the PM. We will discuss these non-canonical assemblies and their functions next.

## Flat clathrin-coated plaques

Flat clathrin lattices have been observed in several cell types and are thought to represent the initial step preceding budding into canonical CCPs in specific cell types, such as HeLa, Cos7 and BSC-1 cells (Avinoam et al., 2015; Lampe et al., 2016; Moulay et al., 2020; Nawara et al., 2022; Sochacki et al., 2021). In addition, clathrin can form large, flat and stable structures, often referred to as clathrin-coated plaques (see poster) (Saffarian et al., 2009). These plaques are abundant in skeletal muscle myotubes and osteoblasts, where they are proposed to contribute to cell adhesion (Akisaka et al., 2003; Grove et al., 2014; Maupin and Pollard, 1983; Vassilopoulos et al., 2014). The presence of a developmentally regulated exon of the CHC was shown to potentiate flat polymerization of clathrin, leading to the formation of extensive plaques in skeletal muscle (Moulay et al., 2020). Interestingly, flat clathrin plaques are also present at enteropathogenic *Escherichia coli* pedestals (Veiga et al., 2007). These non-endocytic, pathogen-triggered structures, which support bacteria on the surface of epithelial cells, take advantage of the structural and organizing properties of the clathrin machinery. Although the precise mechanisms involved in the recruitment of clathrin by bacteria and the function of these clathrin plaques in enteropathogenic pedestals are not fully understood, clathrin plaques and associated branched actin filaments seem to facilitate adhesion of these extracellular bacteria at the cell surface. For an in-depth review of clathrin-coated plaques, see Lampe et al. (2016).
Although the mechanisms controlling plaque formation are not completely understood, important determinants of their assembly have been recently identified. For instance, the αvβ5 integrin, also known as the vitronectin receptor, has been shown to localize in extensive clathrin plaques that form during muscle differentiation, and is suggested to contribute to adhesion during muscle fiber formation (De Deyne et al., 1998; Vassilopoulos et al., 2014). αvβ5 integrin can shuttle between two distinct adhesion structures – focal adhesions (FAs) and flat clathrin lattices (Baschieri et al., 2018; Zuidema et al., 2018). Interestingly, Zuidema et al. found that the localization of αvβ5 integrin depends on cell tension and actomyosin contractility levels; αvβ5 was shown to cluster to FAs under increased cellular tension whereas it redistributed to flat lattices when contractility was lowered. αvβ5 integrin is also involved in the formation of an adhesive reticular network at the plasma membrane of mitotic cells (Lock et al., 2018). Reticular adhesions (RAs) are a recently described type of integrin-based cell adhesion that contain integrin αvβ5, lack typical focal adhesion markers, such as talin1 or paxillin, and are independent of actin stress fibers (Lock et al., 2018). They were found to dominate the adhesion of cells in long-term culture and during mitosis. RAs, as defined by the distribution pattern of αvβ5, often overlap with clathrin-coated plaques, but the extent of the overlap depends on the cell type. RAs are not only found in mitotic cells, but also in interphase cells in which they are assembled at flat clathrin lattices in a process controlled by fibronectin (FN) and its receptor, integrin α5β1 (Hakanpää et al., 2023).

Clathrin plaques are also proposed to function as dynamic actin-controlled hubs for both endocytosis and signaling. In HEK293 and HeLa cells, discrete, diffraction-limited scission events have been detected at both punctate CCPs and at the edges of large, persistent clathrin plaques (Lampe et al., 2016). Using super-resolution microscopy, it has been shown that clathrin plaques associate with both CCPs and actin filaments, suggesting that these sites have endocytic activity (Leyton-Puig et al., 2017). In myotubes, clathrin plaques have been shown to intimately interact with the branched actin nests that surround them and to anchor intermediate filaments (Vassilopoulos et al., 2014; Franck et al., 2019). More recently, the Svitkina group proposed a new model termed ‘edge-pushing’, in which branched actin polymerization promotes invagination of CCPs from flat plaques by pushing at the plaque boundary, thus releasing forces opposing the intrinsic curvature of clathrin lattices. In some instances, these branched actin networks originated from microtubule tips and extended towards CCSs of various morphologies, including spherical and flat lattices (Yang et al., 2022).

Finally, clathrin plaques have been shown to assemble as a function of increasing substrate stiffness, independently of FAs or actomyosin activity (Baschieri et al., 2018). Those authors reported that clathrin plaques act as signaling platforms involved in controlling ERK activity and promoting cell proliferation in stiff environments, and concluded that plaques are mechanotransducing structures that can sense substrate rigidity independently of cell contractility.

## Flat bilayered clathrin–Hrs coats on endosomes

EM was equally instrumental for the discovery of yet another type of clathrin assembly that can polymerize on the cytoplasmic side of the limiting membrane of late multivesicular endosomes (Raiborg et al., 2002; Sachse et al., 2002). These assemblies are termed ‘bilayered clathrin coats’ because of their characteristic stronger electron density in comparison to canonical CCPs when viewed by EM,

which suggests the presence of two (or more) protein layers. The mechanisms of assembly and functions of these particular structures are still largely elusive, but it has been proposed that they might serve as receptor sorting platforms (Raiborg et al., 2002). Endocytosed receptors that are destined for degradation in lysosomes are ubiquitylated and recognized by sorting complexes on endosome membranes. The ubiquitin-binding sorting component Hrs, as well as ubiquitylated cargoes, are enriched in a characteristic flat clathrin coat on the endosome membrane (see poster). Clathrin is required for the clustering of Hrs, which contains a clathrin-box motif (Raiborg et al., 2006). In line with these findings, it has recently been shown that clathrin is required for optimal formation of intraluminal vesicles in multivesicular endosomes, as well as for the sorting of cargos destined to these intraluminal vesicles (Wenzel et al., 2018). In this study, cargo sorting and intraluminal vesicle formation were shown to occur via concerted and coordinated recruitment waves of clathrin and individual endosomal sorting complexes required for transport (ESCRT) subcomplexes on the endosome limiting membrane, and clathrin facilitated the dissociation of ESCRT-0 and ESCRT-I for efficient intraluminal vesicle formation. Thus, flat clathrin lattices not only form on the cytoplasmic side of the PM but also on the cytoplasmic side of intracellular organelles. In both cases, they serve as sorting platforms to concentrate specific receptors, as signaling platforms to transmit chemical and physical cues and as adhesion platforms to provide anchoring points for the cytoskeleton.

## Tubular clathrin–AP-2 lattices

In migrating cells, clathrin and AP-2 form tubular assemblies termed tubular clathrin–AP-2 lattices (TCALs) at collagen-fiber–PM contact sites. Platinum replica EM analyses have revealed these CCSs have a tubular morphology and they wrap around and pinch collagen fibers (see poster) (Elkhatib et al., 2017). TCALs thus provide the cell with anchoring points within 3D extracellular matrix networks. Interestingly, the clathrin adaptor proteins AP-2 and Dab2 accumulate at collagen fiber sites independently of the presence of clathrin. TCAL formation is influenced by collagen fiber-associated growth factors, such as epidermal growth factor receptor (EGFR), which interact with cargoes of the clathrin-mediated endocytosis pathway and increase the nucleation rate of the tubular structures (Bresteau et al., 2021). Tubular lattices are enriched in β1 integrin, which is required for their assembly, and they have been observed to contribute to cancer cell migration in 3D conditions (Elkhatib et al., 2017). Recently, TCALs enriched in β5 integrin have also been shown to pinch, and thus facilitate adhesion, of cancer cells to tubular membranous material deposited onto the substrate by migrating fibroblasts (Baschieri et al., 2023). Thus, beyond clathrin-coated plaques, it appears that additional CCSs have roles in mediating adhesive functions.

## Conclusions and future perspectives

Although CCPs were identified more than 60 years ago, many questions about clathrin biology still remain unsolved. The subsequent discovery of additional non-canonical CCSs, such as flat plaques, bilayered coats and tubular lattices with functions beyond clathrin-mediated endocytosis at the cell surface, underlines the importance of understanding the plasticity of clathrin structures and their roles in signal transduction, cell adhesion and cell–matrix interactions. Although light and electron microscopy have shed light on important aspects of both the structural and functional diversity of clathrin assemblies, the genetic factors responsible for such plasticity have not yet been uncovered. In addition, many
aspects of the mechanisms that drive specific recruitment of clathrin to intracellular TGNs or endosomal membranes remain unknown. Going forward, advances in super-resolution light microscopy and correlative EM techniques combined with advances in single-cell genetic engineering will help to decipher how conventional and unconventional clathrin structures interact and respond to changes in the genetic and physical environment.

### Acknowledgements

We would like to thank Jeanne Lainé and Graça Raposo for contributing original unpublished electron micrographs and Christophe Leterrier/NeuroCyto lab for providing cultured hippocampal neurons. Core funding for our work was provided by the Gustave Roussy Institute, Sorbonne Université, INSERM, and Association Institut de Myologie.

### Competing interests

The authors declare no competing or financial interests.

### Funding

We would like to acknowledge funding from the Agence Nationale de la Recherche (ANR-20-CE13-0024-01, ANR-21-CE13-0018-01 to S.V and ANR-15-CE15-0005-03 to G.M), Institut National du Cancer (2018-1-PL BIO-02-IGR-1) and Fondation pour la Recherche Médicale (DEQ20180339205) to G.M.

### High-resolution poster and poster panels

A high-resolution version of the poster and individual poster panels are available for downloading at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.261674#supplementary-data.

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